Method for diagnosing acute cardiac ischemia

ABSTRACT

Chemiluminescent detection of metabolic by-products of inosine and hypoxanthine is used to diagnose ischemic events such as early acute cardiac ischemia.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention generally relates to the early detection of ischemicevents such as cardiac ischemia. In particular, the invention providesmethods for detecting inosine and/or hypoxanthine, or metabolicby-products thereof, as early biomarkers of ischemia.

2. Background of the Invention

Cardiovascular diseases (e.g. acute myocardial infarction (MI)) are theleading cause of mortality in the world [Naudziunas et al., 2005;Okrainec et al., 2004; Dorner et al., 2004, AHRQ, 2000]. Each year inthe US, approximately 7-8 million patients present with non-traumaticchest pain and seek emergency medical treatment [Morrow et al., 2007].Current emergency medical evaluation on these patients suspected ofhaving acute MI includes obtaining patient history, signs and symptoms,vitals, electrocardiogram (ECG) and blood evaluation for specificcardiac biomarkers [Beyerle, 2002; A.D.A.M. Inc., 2005; Lees, 2000].However, the percent diagnostic accuracy of acute MI when using patientsigns and symptoms, ECG and c-troponin is only approximately 50%. Withthe addition of the recently FDA cleared albumin cobalt binding assay,the diagnostic accuracy improves to approximately 70%; hence the needfor additional research for biomarkers of acute cardiac ischemia tofurther improve patient diagnostic accuracy is important.

The hospital emergency department blood evaluation determines levels ofseveral specific endogenous cardiac protein biomarkers (e.g. cardiactroponin I and T (cTnI, cTnT), creatine kinase-MB (CK-MB) isoform, andmyoglobin). However, these protein biomarkers are indicative of cardiactissue necrosis, and are typically detected hours after the acutecardiac event (infarct), and not at the time of acute cardiac ischemia.

One recent published scientific editorial requested the need for earlyonset biomarkers of acute cardiac ischemia prior to cardiac tissuenecrosis [Morrow et al., 2003]. Ideally, these early onset biomarkerswould aid emergency medical services (EMS) personnel in the rapiddiagnosis and treatment of initial acute cardiac ischemia (potentiallyacute MI), thus increasing the survival rate of acute MI victims everyyear. One research group [Bhagavan et al., 2003] addressing thescientific editorial request, describes a blood measurement for ischemiamodified albumin (IMA), which appears at an elevated level in thebloodstream from patients undergoing an ischemic cardiac event; howeverthe author's state that the colorimetric test would not discriminatebetween cardiac ischemic patients with and without acute MI (e.g.angina), and recent clinical evaluations of the test assay have reportedsignificant false positive results.

This technology is described in U.S. Pat. No. 7,282,369 to Par-Or et al.(Oct. 16, 2007) which teaches rapid methods for the detection ofischemic states and kits for use in such methods. The methods are basedon detecting and quantifying the existence of an alteration of the serumprotein albumin which occurs following an ischemic event. Methods fordetecting and quantifying this alteration include evaluating andquantifying the cobalt binding capacity of circulating albumin, analysisand measurement of the ability of serum albumin to bind exogenouscobalt, detection and measurement of the presence of endogenous copperin a purified albumin sample and use of an immunological assay specificto the altered form of serum albumin which occurs following an ischemicevent. Also taught is the detection and measurement of an ischemic eventby measuring albumin N-terminal derivatives that arise following anischemic event, including truncated albumin species lacking one to fourN-terminal amino acids or albumin with an acetylated N-terminal Aspresidue.

U.S. Pat. No. 7,063,782 to Wayment et al (Jun. 20, 2006) teacheselectrochemical methods and devices for in vitro detection of anischemic event in a patient sample. Following addition of a known amountof a transition metal ion to the patient sample, electrodes are used tomeasure the current or potential difference of non-sequesteredtransition metal ion in the sample. The amount of non-sequesteredtransition metal ion in the sample reflects the degree of modificationto albumin that is the result of an ischemic event. However, severalclinical studies have reported the test to have significant falsepositive results.

There is an ongoing need to discover and develop methods for detectingearly onset biomarkers of acute cardiac ischemia.

SUMMARY OF THE INVENTION

The present invention is based on the development of methods to rapidlydiagnose whether a patient has or is experiencing an ischemic event bydetecting metabolic by-products of xanthine oxidase (XO) activity in abiological sample from the patient. The method is especially useful inemergency situations where a correct diagnosis may be a matter of lifeor death, and must be made as quickly and accurately as possible. Themethods provide medical practitioners (especially emergency medicalpersonnel) with the ability to rapidly distinguish, for example, earlycardiac ischemia from other possible causes of chest pain which havefewer serious immediate consequences (e.g. anxiety, heart burn, etc.),and thus fosters early, appropriate treatment of patients. In addition,the methods may be used to monitor, on an ongoing basis, cardiacpatients or persons considered to be at risk of experiencing orundergoing an adverse ischemic event to insure early detection andintervention. In one embodiment, a chemiluminescent method is used todetect metabolic by-products of XO activity, for example, hydrogenperoxide (H₂O₂) and/or superoxide anion radicals. These products may begenerated by the action of XO on the substrates hypoxanthine and/orxanthine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic drawing of cardiac cellular adenosine triphosphate(ATP) catabolism due to oxidative stress and potential oxygenreperfusion injury due to free radical generation.

FIG. 2. A, High-performance liquid chromatographic diode array detection(HPLC-DAD) chromatograms overlay of control (025-2501.D) and 20-minglobal cardiac ischemia (026-2601.D) mouse perfusate samples. Inosine(retention time/5.9 min) and high-performance liquidchromatography-electrospray ionization-mass spectrometry (HPLC-ESI/MS)identifying inosine (MW/268 Da) as an early biomarker of global cardiacischemia is demonstrated in the ischemic mouse heart perfusate.

FIG. 3. Profile of adenosine triphosphate (ATP) catabolism by-productsfound in Krebs solution versus reperfusion time after 20-min mouseglobal cardiac ischemia. Inosine (μg ml⁻¹) levels were 0, 7.5, 2.1, 1.1,0.4 and not detected for sample time points 0, 1, 3, 5, 10 and 20 min,respectively.

FIG. 4A-E. Chromatograms illustrating (A) 2000 ng/mL hypoxanthine (RT˜5.3 min), uric acid (RT ˜5.8 min), xanthine (RT ˜7.2 min), adenosine(RT ˜10.7 mM) and inosine (RT ˜10.9 min) in deionized water, (B) lowstandard of 250 ng/mL hypoxanthine and inosine in blank plasma, (C)blank plasma, (D) plasma sample from healthy female subject and (E)plasma sample from hospital emergency room female patient.

FIG. 5. Graph of mean percent inosine remaining after plasma PNPmetabolism when stored at 4° C. Square symbols represent fortifiedamounts of 2000 ng/mL of inosine and hypoxanthine in blank plasma (n=3),diamond symbols represent fortified amounts of 250 ng/mL of inosine andhypoxanthine in blank plasma (n=3) and triangle symbols representfortified amount of 2000 ng/mL inosine only in blank plasma (n=3).

FIG. 6. Plot of inosine [μM] to hypoxanthine [μM] (ino/hypo) conversionratio versus reperfusion time (min). The plot represents data from oneanimal heart preparation in group Group IV to 1 mM SA and ischemicexperimental conditions. Ino/hypo conversion ratio is highest in the 1min reperfusion sample and returns to a constant ratio before droppingto zero as aerobic conditions presumably deactivate ADA and PNP enzymesin the cardiac myocytes.

FIG. 7. Diagram of enzymatic conversions of inosine and hypoxanthinecomponents with generation of hydrogen peroxide as a by-product, whichcan react with luminol or lucigenin and HRP to generate visible bluelight (chemiluminescence).

FIG. 8. Diagram of typical reagent addition, injector time points andresulting PHOLASIN® emission (chemiluminescence).

FIG. 9. BMG luminometer set points used for flash mode experiments.

FIG. 10. Typical BMG output luminescence scan for sample analysis of 10μM hypoxanthine in plasma. Range one (background RLU measurement between100 and 120 sec) and range two (peak height RLU measurement between 120sec and 222 sec).

FIG. 11. BMG spread sheet (Excel®) computations, method and dataprocessing set points, and file name are documented for GLP compliance.Results are reported in spread sheet cells based on microplate samplewell location (96 well plates).

FIG. 12. Chart of relative luminescence units (RLUs) versus time (sec)for 30 μM xanthine/XO plate mode kinetics. The profile demonstratessuccessful equipment setup and operation using a commercial test kit forantioxidant evaluation (ABEL 61-M, Knight Scientific). Two individualsamples overlay with analysis time ˜30 min.

FIG. 13. Chart demonstrating a significantly reduced analysis time byutilizing increased amounts of XO (from ˜10.3 mU/ml to ˜676 mU/ml) andcontinuous microplate mixing. Analysis time ˜3.7 min.

FIG. 14A-B. Charts depicting inosine and PNP incubation time andconversion study. A, evaluation of 60 and 120 sec PNP incubation times,with 120 sec demonstrating the complete conversion of inosine tohypoxanthine. B, the 10 μM inosine with PNP conversion RLU responses(n=2) overlays completely against the 10 μM hypoxanthine standard.

FIG. 15A-B. Charts demonstrating effects of uric acid (humanphysiological levels) on PHOLASIN® luminescence signal. A, high levelsof uric acid (in buffer) can quench the luminescence by more than 50%.B, treatment of plasma (1:100 dilution) and use of strong anion exchange(SAX) can reduce antioxidant effect on the luminescence signal andincrease method sensitivity.

FIG. 16. Chart demonstrating the effect of uricase on basal uric acidlevels (normal healthy individual) and with fortification of 10 μMhypoxanthine. The generation of hydrogen peroxide (by-product) fromuricase enzymatic conversion of uric acid to allantoin caused XOinactivity (potentially from hydrogen peroxide product inhibition on XOeffect).

FIG. 17A-B. Charts demonstrating healthy normal individuals and patientswith confirmed acute MI (hospital documented elevated levels of cTnT).A, all cTnT patient samples RLU response were clearly above thecalculated 99% cut-off reference value (5,944 RLU) for healthy normalindividuals (n=6 for each group). B, HPLC values for total hypoxanthineand cTnT values (from ProMedDx) are listed in the legend.

FIG. 18A-B. A, hypoxanthine standard curve in assay buffer ranging from2.3 to 30.3 μM demonstrating sufficient linearity and B, back-calculatedhypoxanthine concentrations.

FIG. 19A-B. Charts demonstrating repeatability of the luminescenceassay. Healthy normal individual (basal level, ˜0.5 μM hypoxanthine) andfortified sample (1.5 μM hypoxanthine) assayed three consecutive times.Overlay of profiles demonstrate plasma sample repeatability.

FIG. 20. Schematic representation of the assay integrated within thediagnosis of a cardiac ischemic event.

FIG. 21. Schematic of competitive binding immunoassay.

FIG. 22. Schematic of enzyme-immunoassay.

FIG. 23A-B. Schematic of point of use hand held device. A, flow chart orprocedure; B, schematic representation of device.

DETAILED DESCRIPTION

The present invention is based on the development of methods to rapidlydiagnose the occurrence of an ischemic event in a patient, by detecting,in a biological sample from the patient, the metabolic by-products (e.g.various reactive oxygen species, ROS) generated by the activity of theenzyme xanthine oxidase (XO). For example, the method is especiallyuseful to rapidly distinguish, at an early stage, acute, cardiacischemia from other possible conditions in a patient who is experiencingchest pain, thus allowing appropriate follow-up treatment. The method isparticularly valuable in emergency situations where emergency personnelotherwise, when using less rapid and less reliable prior art methods,lose valuable time in diagnosing or even misdiagnosing conditions suchas cardiac ischemia.

In one embodiment, the by-products that are detected are one or both ofH₂O₂ and superoxide anion radical (O²—, SAR). These by-products aretypically generated by the action of XO on one or more of the substrateshypoxanthine and xanthine. XO converts hypoxanthine to xanthine andxanthine to uric acid, with concomitant generation of by-products H₂O₂and SAR. The by-products may be generated by exposing a biologicalsample to XO. Alternatively, in some embodiments, the biological sampleis first exposed to the enzyme purine nucleoside phosphorylase (PNP) inorder to convert inosine in the sample to hypoxanthine. Subsequentexposure of the sample to XO generates H₂O₂ and SAR. In the firstembodiment, which does not utilized PNP, XO acts on endogenoushypoxanthine and xanthine (hypoxanthine and xanthine that are alreadypresent in the sample). In the second embodiment, XO acts on both theendogenous hypoxanthine and xanthine, as well as on the hypoxanthinethat is produced by the action of PNP.

The biological samples that are obtained and tested according to themethod may be any that contain elevated levels of inosine and/orxanthine and/or hypoxanthine as a result of ischemia and one or more orall of these substances may be measured. Examples of such biologicalsamples include but are not limited to biological fluids such as blood,plasma, saliva, spinal or brain fluid, breath or aerosol samples, urine,etc.

The ischemic event that is detected may be or be due to a variety ofconditions such as cardiac ischemia, stroke, stable and unstable angina,acute coronary syndrome, and other conditions that are known to beassociated with ischemia.

In order to detect XO metabolic by-products, in some embodiments of theinvention, chemiluminescent methods are used while in other embodimentsof the invention, immunological methods are employed. Depending on theembodiment of the assay that is employed, the sensitivity of the assayis at least in the μM range, and may extend to the nM or even pM rangefor detecting the by-products.

Chemiluminescent Methods

In one embodiment of the invention, the methods involve the use ofchemiluminescence to indirectly detect inosine and/or hypoxanthine bymeasuring the level of one or more metabolic by-products of inosineand/or hypoxanthine. By “metabolic by-products” we mean substances,compounds or other chemical entities that are produced during a chemicalreaction in which inosine and/or hypoxanthine are enzyme substrates forthe enzyme XO. Herein, “metabolic by-products” may also be referred toas “by-products of enzyme reactions”, “metabolites”, “by-products” orother similar terms or phrases that are recognized by those of skill inthe art. In one embodiment, the metabolites are by-products of thecatalysis of hypoxanthine by the enzyme xanthine oxidase (XO). Inanother embodiment, the metabolites are by-products of the catalysis ofinosine and hypoxanthine by the enzymes purine nucleoside phosphorylase(PNP) and xanthine oxidase (XO). Biological enzymes PNP and XO arespecific for enzymatic conversions of inosine and hypoxanthine,respectively. The PNP enzyme converts inosine to hypoxanthine and XOconverts hypoxanthine to xanthine, followed by XO conversion of xanthineto final product uric acid (in human species). Each time XO reacts withone mole of hypoxanthine and with one mole of xanthine, the metabolicby-products of each XO enzymatic turnover is the production of one moleof hydrogen peroxide (H₂O₂) and two moles of superoxide anion radical(O₂ ⁻.). Both of these by-products can become substrates forluminescence (e.g. chemiluminescence) type reactions and the presentinvention is based on the development of rapid and accuratechemiluminescent assays that detect and measure the level (quantity,amount, etc.) of one or both of H₂O₂ and (O₂ ⁻.). The by-products mayalso be substrates for colorimetric, phosphorescent or fluorescent typereactions that allow for measurable detection.

In general, the methods of the invention involve obtaining a blood orplasma sample from a patient that is or has recently experienced chestpain. Typically, a blood sample is obtained using a suitable technique,many of which are known to those of skill in the art. For example,lithium heparin tubes are known and may be used. The plasma component ofwhole blood is separated, e.g. by centrifugation, filtration, etc. orother suitable methods. Typically, a molecular weight cutoff (MWCO)filter in the range of 5,000 to 50,000 is sufficient, with a range of10,000 to 35,000 being preferable. Centrifugation at e.g. ˜1000×g forabout 1-10 minutes or less will usually be appropriate, and is wellwithin the purview of one of skill in the art to determine. Suitablealiquots of the plasma sample are then treated according to the methodsof the invention to convert inosine and hypoxanthine in the sample tospecies that are readily detected by chemiluminescence, as follows.

PNP Reaction

In this embodiment of the invention, aliquots of plasma are combinedwith PNP, e.g. the plasma may be transferred into a suitable reactionvessel and an appropriate quantity of PNP is then added to the reactionvessel, or plasma may be added to a reaction vessel that alreadycontains PNP. In either case, the combining/mixing of the plasma and PNPis carried out under conditions that allow the PNP to convert inosine inthe sample to hypoxanthine. Suitable reaction vessels include but arenot limited to, for example, plates containing wells such as 96-wellplates, various known glass and plastic test tubes, by spotting thereaction mixture onto a substrate, within various known hollowmicrofibers, etc. In a preferred embodiment, the reaction vessel is a96-well plate. Generally, the volume of plasma that is utilized is inthe range of from about 1 μl to about 200 μl, and preferably is about 20μl. In addition, the plasma aliquot may be diluted as well, e.g.appropriate media may be in the reaction vessel and the plasma added toit, or added to the reaction vessel after the plasma had been added.Generally, the amount of dilution will be in the range of from about 0to about 100-fold, or from about 0 to about 50 fold, or from about 0 toabout 25-fold, or from about 0 to about 10-fold. Suitable dilutionbuffers include any that allow the enzyme reactions to proceed at asufficient rate (e.g. at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100% or possibly even more) of the rate that is displayedunder optimal standardized conditions, so as to achieve detectableamounts of product within the rapid time frame of the assay. At the sametime, the buffers utilized in the assay must not interfere with thechemiluminescence that is generated e.g. they must permit at least about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 100% of the maximalpossible luminescence. Generally, these two objectives can be achievedby using buffers which buffer in a pH range of from about 7.0 to 8.0, orpreferably from about 7.2 to about 7.8, and more preferably at or aboutpH 7.4. In other words, the buffers that are utilized buffer at or nearphysiological pH, as understood by those of skill in the art. Buffers ofchoice include but are not limited to, for example, various phosphatebuffers (e.g. dibasic sodium phosphate, potassium phosphate, etc.);various Tris buffers that buffer at or near pH 7.4; Zwitterionic“Good's” buffers that buffer at or near pH 7.4; borate buffers;imidazole buffers, amino acid containing buffers (e.g. histidine), andothers that will occur to those of skill in the art. In a preferredembodiment, the buffer is 20 mM dibasic sodium phosphate, pH 7.4.

As will be understood by those of skill in the art, to minimizeexperimental variability, each assay preferably is carried out at leastin duplicate, possibly triplicate, or with even higher numbers ofidentical repetitive aliquots. PNP enzyme is added to each reaction toachieve a final concentration that is generally in the range of fromabout 100 to about 1000 mU/ml, or from about 200 to about 900 mU/ml, orfrom about 300 to about 700 mU/ml, typically from a standardized stocksolution. Those of skill in the art will recognize that the amount ofenzyme that is used may be varied and/or optimized depending on severalfactors (e.g. temperature, volume, method of detection, etc.). The PNPis incubated in the reaction vessel with the plasma under conditionsthat are favorable for quantitative conversion of inosine tohypoxanthine, e.g. at a constant temperature in the range of from about20° C. to about 40° C., and preferably in the range of from about 25° C.to about 37° C., and most preferably at 25° C. or at 37° C. Generally,the incubation reaction is allowed to proceed for a time period in therange of from about 60 seconds to about 5 minutes or less. Conversion istypically complete after about 120 seconds of incubation. Although thoseof skill in the art will recognize that longer incubations may be usedif desired, in the interest of reducing the time required for this rapidassay, while still achieving quantitative conversion of the substrates,a preferred reaction time is 120 seconds.

Alternatively, PNP may also be added to the sample during thecentrifugation (plasma separation) step in order to further speed theprocedure. For example, a sufficient quantity of PNP may be addeddirectly to the blood sample before centrifugation; or PNP may be addedto plasma before the plasma is transferred to the reaction vessel, orthe sample tube used for collecting the blood sample may be coated orpartially coated with PNP, etc. Any suitable strategy for combining theplasma sample with PNP or for contacting the sample with PNP may beused, so long as conditions are such that inosine in the plasma issubstantially converted to hypoxanthine. Examples include but are notlimited to immobilizing the enzyme on a substrate (e.g. beads, strips,etc.), or even spotting (placing or otherwise transferring) the sampleonto a suitable substrate and exposing the substrate, to a solution ofenzyme, etc.

The PNP enzyme that is utilized may be from any suitable source. In someembodiments, the PNP is human PNP. In other embodiments, other sourcesare used, e.g. bacterial PNP.

XO Reaction

Thereafter, (i.e. preferably after about 120 seconds) a suitablequantity of XO enzyme is mixed with the reacted sample to effect thequantitative conversion of hypoxanthine to xanthine. In mostembodiments, XO is added directly to the plasma/PNP mixture in thereaction vessel. However, as discussed above, in some embodiments, PNPis not used and the blood or plasma sample is contacted with XO toconvert endogenous hypoxanthine and xanthine. XO enzyme is added to eachreaction to achieve a final concentration that is generally in the rangeof from about 100 to about 1000 mU/ml, or from about 200 to about 900mU/ml, or from about 300 to about 700 mU/ml. Those of skill in the artwill recognize that the amount of enzyme that is used may be variedand/or optimized depending on several factors (e.g. temperature, volume,method of detection, etc.). The XO is incubated in the reaction vesselwith the plasma under conditions that are favorable for quantitativeconversion of hypoxanthine to xanthine and xanthine to uric acid, e.g.at a constant temperature in the range of from about 20° C. to about 40°C., and preferably in the range of from about 25° C. to about 37° C.,and most preferably at 25° C. or at 37° C.

Generally, production of by-products begins immediately upon theaddition of XO. Monitoring of the production of by-products may beginany time after addition of XO, and preferably immediately after, sincethe reaction is substantially over after about 30 seconds. Theluminescent signal may be monitored throughout the reaction, or may bemonitored at one or more selected time intervals.

Those of skill in the art will further understand that rather than“adding” XO to the sample, the sample may be contacted with XO by someother means, e.g. XO may be immobilized on beads, strips, or othersubstrates, etc., and the sample may be brought into contact with theenzyme by an appropriate means, e.g. dipping or spotting, etc. Inaddition, the sample may be spotted or otherwise located or placed ontoa suitable substrate and the substrate may then be exposed to a solutionof enzyme, etc. Further, in some embodiments, both PNP and XO may beco-located on a suitable substrate.

Chemiluminescent Measurement

As will be understood by those of skill in the art, the precise mannerin which by-product measurement is carried out depends on whichby-products (H₂O₂ or SAR, or both) are measured, and which reagent isused to detect the by-product(s). Detection reagents are typically addedto the reaction mixture either together with or before the addition ofXO and, as discussed above, the reaction is monitored immediately.

In one embodiment of the invention, SAR are detected. Since one mole ofhypoxanthine generates 4 moles of superoxide anion radicals (SAR) as aby-product of XO activity, using a chemiluminescent material that reactswith SAR should theoretically provide more luminescence signal thanwould H₂O₂, potentially increasing the sensitivity two fold incomparison. Thus, when SARs are measured, very high sensitivity isachieved even at low concentrations or inosine and hypoxanthine in theblood sample. Examples of reagents that can be used in the detection ofSAR include but are not limited to lucigenin (bis-N-methylacridinium);PHOLASIN®; and others that may occur to those of skill in the art.PHOLASIN® utilizes the highly sensitive bioluminescent photoproteinPHOLASIN® from the bivalve mollusc Pholas dactylus. In a preferredembodiment, PHOLASIN® is used.

However, in other embodiments of the invention, H₂O₂ may be detected. Inthis case, reagents including but not limited to horseradishperoxidase/lucigenin, horseradish peroxidase/luminol, etc. may beutilized. If using luminol or lucigenin as the luminescent material, thehydrogen peroxide (which has both oxidizing and reducing capabilities)reacts with the horseradish peroxidase (HRP) (or other peroxidase)enzyme to generate hydroxyl radical, which in turn reacts with luminolto generate measurable blue light ˜450 nm. Thus an amplification of thesignal (one mole of hypoxanthine and xanthine generates two moles ofhydrogen peroxide) occurs. Preferably, the reaction is also carried outwith signal enhancers.

Signal enhancers may be added to any of the luminescence reactionsdescribed herein to increase the detectable signal produced by reactionof the by-product with a chemiluminescent reagent. Examples of signal(sensitivity) enhancers include but are not limited to Adjuvant-K™(which is specific for PHOLASIN®), and other that may occur to those ofskill in the art. The use of an enhancer may be especially importantsince this is a rapid assay and does not generally require “clean-up” ofthe sample prior to analysis. Therefore, substances that interfere withthe enzyme reactions or with the luminescent labeling or signal maystill be in the sample. In other embodiments of the invention, some“clean-up” techniques may be employed, which include but are not limitedto strong anion exchange (SAX) for removal of organic acids, other solidphase extractions (e.g. silica), the addition of base to salt outorganic acids, various liquid/liquid extractions, etc.

In other embodiments of the invention, more than one by-product isdetected. For example, both H₂O₂ and SARs may both be detected, eitherin the same reaction mixture, or in parallel side-by-side reactions, oneof which measures H₂O₂ and the other of which measures SAR.

Typically, the chemiluminescent reagent's reaction with a by-product isdetected at a characteristic wavelength, usually a wavelength at whichthe signal is maximal, or at which other substances in the reaction donot interfere significantly. Those of skill in the art are familiar withobtaining such measurements. Generally a suitable automated luminometeris utilized as described in Example 4 below, and any such means formeasuring a suitable wavelength of light produced in proportion to theabout of by-product(s) of interest may be used in the practice of theinvention.

Time Required for Assay

The assay of the present invention is a rapid assay, i.e. it cangenerally be completed in about 10 minutes or less, after a blood sampleis obtained from the patient. Those of skill in the art will realizethat the time frame of the assay does not take into account transportingthe sample to a lab where the analysis takes place, but assumes that thetime begins one the blood sample is in the hand of a skilledprofessional, i.e. one who is trained to carry out the assay. Likewise,the end point of the assay is considered to be the time as which datacan be read out from the instrument that is used to measureluminescence. In some embodiments of the invention, the time requiredfor the assay is about 10 minutes or less, 9 minutes or less, 8 minutesor less, 7 minutes or less, 6 minutes or less, 5 minutes or less, 4minutes or less, 3 minutes or less, 2 minutes or less, or even 1 minuteor less.

Interpreting the Results

Those of skill in the art are familiar with the use of baselines orcontrols for determining the significance of a measurement, and withtechniques for calibrating an instrument, and it is understood that suchappropriate measures would be taken to ensure accuracy andreproducibility of the assay. Typically, measurement of a level ofby-product in a patient sample that exceeds a 95%, 96%, 97%, 98%, 99% oreven higher (e.g. 99.9%), and preferably a 99% or higher confidenceinterval threshold, when compared to normal controls, is considered tobe a positive indicator of cardiac ischemia. The cutoff threshold istypically 4.6 μM combined concentration of inosine, xanthine, andhypoxanthine, which corresponds to a RLU reading in the range of about180,000, depending on the instrument that is used to carry out themeasurement and the calibration of the instrument. Those of skill in theart will recognize that RLU quantities may vary, depending on theinstrument, its calibration, etc. Conversely, levels below thisthreshold indicate that the patient has not yet experienced cardiacischemia. Such evaluations will typically be made by a skilledprofessional such as a physician, physician's assistant, trainedemergency worker, etc. However, health care professionals will alsousually prefer to repeat the test after a suitable length of time toconfirm the initial finding. The test may be repeated one or more times,as necessary, until the health care professional is satisfied that acorrect diagnosis has been made, and appropriate action has been taken.

FIG. 20 depicts a representative scenario for intake procedures andtriage of a patient that presents with chest pain, and Example 4 furtherdiscusses this aspect of the invention. In addition to taking vitalsigns and history, a panel of known cardiac tests are carried out,including the inosine and/or hypoxanthine test described herein. If thelevels of inosine/hypoxanthine are elevated, appropriate interventionimmediately ensues and the patient is treated as though cardiac ischemiais occurring, has occurred or is imminent. The inosine/hypoxanthineassay may be repeated later (e.g. after about 1-3 hours) to confirm theefficacy of intervention, and thereafter as necessary to insure propertreatment of the patient.

On the other hand, if the initial test indicates theinosine/hypoxanthine levels are not elevated, the patient may simply beobserved and/or other tests may be carried out to determine if there isanother cause of the chest pain. The inosine/hypoxanthine test may berepeated (e.g. after about 1-3 hours) in order to confirm the initialresult, at which time a decision will again be made regarding whether ornot cardiac ischemia is indicated. If yes, then appropriate treatmentensues. If not, then ischemia may be ruled out and an alternate suitabletreatment protocol is prescribed.

2. Immunological Detection of Inosine and/or Hypoxanthine

In another embodiment of the invention, the method to detect inosineand/or hypoxanthine in immunological in nature and involves the use ofantibodies specific for one or the other or both of inosine andhypoxanthine. In one embodiment of the invention, the antibodies aremonoclonal antibodies. Immunoassay is a well established sensitivetechnique commonly used in clinical chemistry environments. Thisembodiment of the invention provides a sensitive immunoassay technique,which utilizes antibodies for detection of inosine and hypoxanthinelevels in biological fluids (e.g. plasma, serum, whole blood). Afterdevelopment of the antibodies, several established quantitativetechniques using antibodies may be utilized (e.g. Competitive Binding,FIG. 21; or a “Sandwich” Assay, FIG. 22).

For development of the antibodies, due to the small size of inosine andhypoxanthine (<300 Daltons molecular weight), these molecules mustgenerally be conjugated to a carrier protein (e.g. albumin) to elicit anantibody response in the host animal (e.g. rabbit, goat, and mouse). Thecurrent established techniques for producing and isolating monoclonalantibodies are preferred over polyclonal antibodies, as this willultimately increase the specificity of the testing assay. The producedantibodies can be tagged (fluorescent label) using current establishedlabeling techniques.

The resulting monoclonal antibody (tagged and untagged) can be utilizedas shown in FIGS. 21 and 22, in a manner similar to that which is usedfor quantitative detection of biomarkers of acute myocardial infarction(e.g. myoglobin, CK-MB, cardiac troponin). The main advantages of usingan immunoassay technique includes sensitivity (fluorescence) andspecificity (antibody) in the detection of inosine and hypoxanthine, andthe potential utilization of the developed antibodies in current cardiacpanel assays (e.g. with myoglobin, cardiac troponin, CK-MB).

The immunoassay technique may have at least two clinical applications.The immunoassay may be added to existing hospital clinical laboratoriesquantitative cardiac panels. Alternatively, or in addition, theantibodies may be used on qualitative cardiac panel test strips. Bothapplications may be useful as a medical diagnostic tool for detection ofacute cardiac ischemia in non-traumatic chest pain (or other) patients.

Point of Care (POC) Device

The invention also comprehends a rapid hand-held medical device forpoint-of-care whole blood levels of endogenous inosine and/orhypoxanthine, potential biomarkers of non-traumatic acute cardiacischemia. The purpose of using the hand held medical device is torapidly measure acute cardiac ischemia biomarkers (e.g.inosine/hypoxanthine) from a finger stick whole blood sample. Theresults of the evaluation should take less than 1 minute, whichsignificantly reduces the time course for emergency medical diagnosisand treatment of individuals experiencing non-traumatic acute cardiacischemia or impending acute myocardial infarction.

The point-of-care medical device comprises hardware (similar to thatutilized in commercially available hand-held glucose meters), software(for meter operations and computations), and disposable substrate (e.g.test strips) with bound enzymes and luminescent material for direct orindirect quantification of inosine/hypoxanthine in whole blood. Thehand-held medical device is similar in basic operation to the commercialhand-held glucose meter used by diabetic patients. The major differencesbetween this invention and the glucose meter are the following: theglucose meter technology utilizes glucose oxidase on the test strips andtypically a potentiometric detector, whereas one embodiment of theinvention utilizes enzymes PNP and XO, PHOLASIN® as the luminescentmaterial (alternate luminescent materials can also be used), andpreferably a photomultiplier tube (PMT), photodiode or an equivalentmeans of detection, as the detector. Luminescence technology issignificantly more sensitive than the potentiometric detection. Purinenucleoside phosphorylase (PNP) and xanthine oxidase (XO), the enzymesused in the disposable test strip, are available commercially (e.g.Sigma-Aldrich, USA). The enzymes (which address enzyme substratespecificity) are covalently bound to a substrate such as a test strip,and used to convert inosine to hypoxanthine, hypoxanthine to xanthine,and xanthine to uric acid. During XO enzymatic activity, superoxideanion free radicals are generated as a by-product of enzyme turnover.PHOLASIN®, a sensitive photoprotein from a bi-valve mollusk, is alsocovalently bound to the test strip and generates visible light in thepresence of superoxide anion free radicals to produce measurableblue-green light (luminescence, ˜490 nm). Luminescence is a sensitivetechnique to provide an indirect measurement (measures componentenzymatic conversion by-products) of inosine and hypoxanthineconcentrations in whole blood. In addition, use of the medical devicemay be coupled to the use of cell phone technology. In this case, ifmeasured whole blood concentrations of inosine/hypoxanthine wereatypically elevated, an automatic notification could be transmitted bythe medical device to activate an Emergency Medical Services system. Therapid measurement of elevated inosine and hypoxanthine in whole bloodshould aide EMS personnel in initiating immediate treatment for acutecardiac ischemia in non-traumatic chest pain patients.

The hand-held medical device is a rapid and quantitative instrumentwhich may be used by hospital emergency department (ED) or EMS personnelas part of the initial medical assessment on patients presenting withnon-traumatic chest pain and potential acute cardiac ischemia.Generally, the device is modular (handheld), automated and easilyoperated by trained individuals (e.g. emergency or other medicalservices personnel, or even by patients themselves). The medical deviceand test strips are affordable for potential use at home by individualswhose physician have classified them as at high risk for developingacute myocardial infarction (e.g. unstable angina, medical history ofmyocardial infarction, etc).

This aspect of the invention is illustrated schematically in FIG. 23A-B,where FIG. 23A is a flow diagram of a procedure that may be followedusing the device, and FIG. 23B is a schematic representation of thedevice 10, which comprises substrate 11 and detector 12.

Detection Using HPLC

In some embodiments of the invention, early biomarkers of ischemia (suchas inosine, xanthine, hypoxanthine, adenine and uric acid) are measureddirectly using chromatography methods such as HPLC. One or more, andpreferably two or more, of these biomarkers may be measured in aclinical sample from a patient in order to determine whether or not thepatient has or is experiencing ischemia. The sample may be exposed toPNP and/or XO enzymes as described above, or the sample may be analyzeddirectly with no enzymic conversion. Those of skill in the art willrecognize that some preparatory steps may be taken to process thesample, e.g. centrifugation, filtration, etc. The output of the analysisis typically a direct quantification of the amount of the detectedsubstances in the sample, based on parallel or corresponding analyses ofcontrol samples containing known quantities of the substances.

Uses of the Methods

The methods of the invention may be advantageously employed in anysetting in which they would be beneficial, and especially in clinical ormedical settings. In particular, any emergency facility (e.g. anemergency room, ambulance, etc.) may employ the methods. In someembodiments, the blood sample that is obtained from the patient isanalyzed in a laboratory setting. However, this need not be the case. Asdiscussed above, the methods of the invention can also be adapted forpoint of care use.

The assay described herein may be carried out in concert with otherassays and/or tests, i.e. the assays of the invention may be part of abattery of tests which are generally used to evaluate patients withnon-traumatic chest pain. However, those of skill in the art willrecognize that in some circumstances persons who have experiencedtraumatic chest pain may also benefit from the use of the methods anddevices, as well as persons who are not experiencing chest pain but maybe at risk of developing cardiac ischemia. In other embodiments, theassay described herein may be combined with one or more other assays(e.g. glucose oxidase/horse radish peroxidase, HRP assay) in a singlereaction, e.g. on a single test strip.

The following examples are intended to illustrate the practice of theinvention but are not intended to limit the scope of the invention inany way.

EXAMPLES Example 1 High-Performance Liquid Chromatography (HPLC)Determination of Inosine, a Potential Biomarker for Initial CardiacIschemia, Using Isolated Mouse Hearts

Each year in the USA approximately 78 million patients withnon-traumatic chest pain come to hospital emergency rooms. It isestimated that approximately 25% of these patients are experiencingcardiac ischemia, but due to the shortcomings of the available testingmethods they are incorrectly diagnosed and discharged withoutappropriate therapy having been provided. Preliminary data with aglobally ischemic mouse heart model has demonstrated that endogenousinosine might be a potential biomarker of initial cardiac ischemiabefore cardiac tissue necrosis. A high-performance liquidchromatographic diode array detection (HPLC-DAD) method was utilized forthe detection and quantification of inosine in Krebs Henseleit (Krebs)buffer solution perfusing from surgically removed and isolated mousehearts undergoing global cardiac ischemia. A C₁₈ column at a flow rateof 0.6 ml min 1 with an aqueous mobile phase of trifluoroacetic acid(0.05% trifluoroacetic acid in deionized water, pH 2.2, v/v) andmethanol gradient was used for component separation. The assay detectionlimit for inosine in Krebs buffer solution was 500 ng ml 1 using a100-ml neat injection. The HPLC results were used to determine totalcardiac effluxed inosine into the Krebs effluent for each mouse duringoxidative stress and compared with the percent cardiac ventricularfunctional recovery rate to determine if a relationship exists amongstthis cardiovascular parameter during periods of cardiac oxidativestress.

Introduction

Cardiovascular disease (e.g. myocardial infarction) is one of theleading causes of mortality in the world (Domer & Rieder 2004, Okrainecet al. 2004, Naudziunas et al. 2005). Current medical evaluation ofpatients suspected of having a myocardial infarction includes anelectrocardiogram blood evaluation for specific biomarkers of cardiacischemia and where available radioisotope perfusion studies (Lees 2000,Beyerle 2002, ADAM, Inc. 2005). Blood evaluation determines the levelsof several specific endogenous protein biomarkers (e.g. troponin T,troponin I, creatine kinase MB (CK-MB) and myoglobin); however, thesebiomarkers are normally indicative of cardiac tissue necrosis and aredetected hours after the cardiac ischemic event and not at the time ofinitial cardiac ischemia, which may include angina (stable or unstablebut non-necrotic). Ideally, emergency medical services would benefitfrom a biomarker of early cardiac ischemia to guide initial treatmentand subsequent diagnostic steps in the chest pain patient. Medicalconditions (e.g. anxiety attacks, acid reflux and angina) other thanmyocardial infarction that cause patient chest pain and otherconstitutional symptoms that might be seen as being consistent withmyocardial ischemia.

To perform its circulatory function, the heart is highly energydependent on adenosine triphosphate (ATP), which is made in cardiaccellular mitochondria by either aerobic (oxidative phosphorylation viaelectron transport chain) or anaerobic (glycolysis) processes. Theaerobic process is heavily oxygen dependent and generates approximately80% of cardiac cellular ATP. The anaerobic process is independent ofoxygen and produces approximately 20% of the cardiac cellular ATP.Lactic acid is a by-product of anaerobic ATP production.

To produce large quantities of ATP, human cardiac cells have anabundance of mitochondria that comprise approximately 40-50% of thecardiac cellular mass. When cardiac tissue is subjected to periods ofconstant oxidative stress (e.g. cardiac ischemia), insufficient oxygenis available for cardiac mitochondria to synthesize aerobically the ATPrequired for normal cardiac function. This causes a cellularaccumulation of ATP metabolic by-products (e.g. adenosine diphosphate(ADP), adenosine monophosphate (AMP)) and activates normally dormantenzymes (e.g. 5′-nucleotidase, adenosine deaminase, purine nucleosidephosphorylase and xanthine oxidase) to catabolize the ATP by-products tosubstances such as adenosine, inosine, hypoxanthine, xanthine and uricacid for cardiac cellular elimination (Abd-Elfattah et al. 2001). Inhuman cardiac tissue, another source of ATP metabolic by-products isthrough metabolism of diadenosine polyphosphates, which are releasedfrom cardiac specific secretory granules during periods of cardiacmetabolic or ischemic stress to provide cellular protective functions(Luo et al. 2004).

Inosine (9-β-D-ribofuranosylhypoxanthine) is an endogenous purinenucleoside normally found in the human body as a degradation componentof purine metabolism. In human plasma, inosine is metabolized in redblood cells with a reported half-life of <5 min with endogenous plasmalevels found in trace amounts (e.g. low ng ml⁻¹) (Viegas et al. 2000).In humans, nature has provided a cellular biochemical mechanism to helpconserve energy in producing the required large quantities of ATP forcardiac cellular use (called the salvage pathway), which can convertcellular inosine back to ATP via several enzymatic steps; thus,recycling cellular inosine (Nelson & Cox 2000). However, in periods ofconstant cardiac oxidative stress (e.g. 20 min), cardiac cells build upsignificant amounts of ATP metabolic by-products, which activatenormally dormant enzymes to catabolize ATP by-products, which thenbecome systemically available before their elimination.

A recently published scientific editorial requested the need for aninitial biomarker for cardiac ischemia before cardiac tissue necrosis(cardiac proteins found in plasma after several hours of cardiacischemia) (Morrow et al. 2003). This initial biomarker would aidEmergency Medical Services (EMS) personnel in the rapid treatment ofinitial cardiac ischemia (potentially myocardial infarction), thuspotentially increasing the survival rate of myocardial infarctionvictims every year. One recent publication (Bhagavan et al. 2003)addressing the scientific editorial request describes a bloodmeasurement for serum albumin that appears at an elevated level in theblood in patients undergoing myocardial infarction. However, the authorsstate that the colorimetric method would not discriminate betweenischemic patients with and without myocardial infarction, thus the needfor a method to detect the initial cardiac ischemic event beforemyocardial infarction would be beneficial to EMS personnel.

Before extracellular biomarkers (e.g. serum albumin) appearing in theblood from cardiac ischemic events, plasma inosine levels would beelevated significantly above the normally low endogenous levels thusbecoming a useful biomarker of pre-necrosis cardiac ischemia. Adenosine,another nucleoside metabolic by-product of ATP catabolism, ismetabolized by red blood cells and has a very short plasma half-life(e.g. approximately 15 seconds); thus making it more difficult tomeasure it quantitatively in plasma (Mei et al. 1996).

The Institute of Cancer Research (ICR) outbred mouse (Dohm 2004) wasused as the animal model for all global cardiac ischemia experimentsusing a Langendorff apparatus (Xi et al. 1998). For sample analysis, ahigh-performance liquid chromatographic diode array detection (HPLC-DAD)method was utilized consisting of direct injection of the Krebs buffereluant from surgically removed and perfused mouse heart tissue. Inaddition, the HPLC-DAD method utilized current column technology(hydrophobic/hydrophilic reverse-phased retention), which providedsufficient component resolution and sensitivity for adenosine, inosineand xanthine-like derivatives. The HPLC-DAD results were used to computethe inosine area under the concentration (AUC) time curve from mouseKrebs buffer eluant samples and compared with the percent cardiacventricular functional recovery rate to determine if a relationshipexist between this cardiovascular parameter during periods of constantcardiac oxidative stress.

Materials and Methods Chemicals, Mobile Phase and Krebs Buffer Solution

Hypoxanthine and xanthine were purchased from Acros Organics (Fair Lawn,N.J., USA). 2,3-Dihydroxybenzoic acid (DHBA), 2,5-dihydroxybenzoic acid,salicylic acid (SA), adenosine, inosine and uric acid were purchasedfrom Sigma-Aldrich (St Louis, Mo., USA). Sodium chloride, sodiumbicarbonate, potassium chloride, magnesium sulfate, monobasic potassiumdihydrogen phosphate, dextrose and calcium chloride were used to preparethe Krebs buffer solution, and all were purchased from Sigma-Aldrich.All purchased chemicals were ACS reagent grade or better. The Krebsbuffer solution (118.5 mM NaCl, 25.0 mM NaHCO₃, 11.1 mM C₆H₆O₆, 4.7 mMKCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄ and 2.5 mM CaCl₂) was prepared inultrapure deionized water at pH 7.4 and with a 95% O₂:5% CO₂ ratio. Formobile phase preparation, trifluoroacetic acid (TFA) was reagent gradeand methanol was Optima HPLC-grade; both were purchased from FisherScientific (Fair Lawn, N.J., USA). Ultrapure distilled and deionizedwater (18 megohm) used for HPLC work was prepared in-house using thePurelab Ultra deionized water system (US Filter, Lowell, Mass., USA) andfiltered before use.

Preparation of Standard Solutions

Stock standards of adenosine, inosine, hypoxanthine, xanthine and uricacid (100 μg ml⁻¹) were prepared in deionized water and stored at 4° C.Working standards of each component were prepared at 2.5 ml⁻¹ in Krebsbuffer solution and maintained at −20° C. along with the mouse Krebsbuffer eluant samples. The working standards stored at −20° C. werestable for at least 6 months.

HPLC-DAD, High-Performance Liquid Chromatography-Mass Spectrometry(HPLC-Ms) Equipment and Conditions

For inosine quantification and diode array spectral purity, the HPLCequipment consisted of an Agilent Model 1100 Quaternary HPLC-DAD andChemstation software (Palo Alto, Calif., USA). The DAD was set toacquire a complete ultraviolet light spectrum for component specificitywith 240 nm used for quantification of inosine and the other ATPmetabolic by-products. For inosine confirmation, liquidchromatography/mass spectrometry (LC/MS) was used and the equipmentconsisted of a Shimadzu LCMS-2010A HPLC coupled to a single quadrupolemass spectrometer using LCMS Solutions software (Columbia, Md., USA).The HPLC-MS conditions consisted of using electrospray ionization (ESI)with the following instrument set points (heating block at 300° C.,nebulizer at 4 liters/per minute nitrogen, interface voltage at 2 kV)and full-scan acquisition using a positive-ion mode.

The analytical column for both HPLC-DAD and HPLC-MS analysis was aSynergi™ Hydro-RP C₁₈, 150×3 mm i.d., 4 mm packing, 80 Å (Phenomenex,Torrance, Calif., USA). The C₁₈ guard column was a 30×4.6 mm i.d., 40-50μm pellicular packing (Alltech, Deerfield, Ill., USA). The mobile phaseconsisted of aqueous trifluoroacetic acid (0.05% TFA in deionized water,v/v, and pH 2.2) and methanol gradient. The mobile phase gradient waslinear with a time-course as follows (95:5 0.05% TFA in deionizedwater:methanol, v/v at 0 min; 70:30 0.05% TFA in deionizedwater:methanol, v/v at 12 min; 10:90 0.05% TFA in deionizedwater:methanol, v/v at 13 min and held 3 min, and 95:5 0.05% TFA indeionized water:methanol, v/v at 17 min).

The mobile phase was degassed automatically using an Agilent 1100membrane degasser with a flow-rate of 0.6 ml min 1. An injection volumeof 100 ml of the Krebs buffer eluant was made using an autosampler. Thetypical HPLC operating pressure was approximately 150 bar with ambientcolumn oven temperature and 345 kPa backpressure regulator (SSI, StateCollege, Pa., USA) to prevent mobile phase outgassing in the detector.

ICR Mouse Experiment Conditions

ICR mice were used for all cardiac ischemia experiments withmorphometric characteristics and baseline cardiac function of the adultmice (ICR strain) provided in Table 1. The mice were anaesthetized;hearts were surgically removed and isolated using the Langendorffapparatus. Global cardiac oxidative stress was accomplished by adjustingthe Krebs-buffered solution to zero flow through the heart for 20 min.Upon heart reperfusion, approximately 1.5-ml samples of Krebs-bufferedeluant from the isolated mouse hearts were collected at predeterminedtime-points (0, 1, 3, 5, 10 and 20 min) in plastic bullet centrifugetubes and frozen at −20° C. until HPLC-DAD analysis.

To evaluate the effects of oxidative stress on the mouse heart,established cardiovascular measurements (e.g. ventricular functionalrecovery) were performed on both control and test animals. Themethodology used to evaluate the isolated perfused mouse heart has beenpreviously described (Xi et al. 1998). In brief, animals wereanaesthetized with an intraperitoneal injection of pentobarbital sodium(100 mg kg⁻¹, with 33 IU heparin added). The heart was removed andimmediately placed in ice-cold Krebs buffer. The aorta was cannulatedwithin 3 min onto the Langendorff perfusion system and the heart wasperfused in a retrograde fashion at a constant pressure of 55 mm Hg withKrebs buffer gassed with 95% O₂ and 5% CO₂. The pH of the buffer and theheart temperature were maintained at 7.35-7.50 and 37±0.5° C.,respectively. A force-displacement transducer (Grass, FT03) was attachedto the apex via a metal hook/surgical thread/pulley system to record andmeasure the ventricular contractile force and heart rate continuously.For each heart the resting tension was set at approximately 0.3 g in thebeginning of the experiment.

The protocol for the test group consisted of 30 min of stabilization, 20min of zeroflow global ischemia, and 30 min of reperfusion (Xi et al.1998). Time-matched normoxic perfusion was carried out for the controlgroup. At the end of each experiment, the heart was removed from theLangendorff system, quickly weighed and stored at −20° C.

Sample Preparation, Stability and Instrument Precision Evaluation

Before HPLC analysis, perfusate samples frozen at −20° C. were thawed toambient temperature, mixed thoroughly by inversion and transferred toplastic autosampler vials for subsequent direct injection into theHPLC-DAD system. To evaluate sample stability in the perfusate solutionand instrument precision; prepared samples in autosampler vials werestored at ambient laboratory temperature overnight and reinjected (n=3times) into the HPLC for analysis.

TABLE 1 Morphometric characteristics and baseline cardiac function ofthe adult mice (ICR strain). Ischemia reperfusion Control (n/6) test(n/6) Body weight (g) 42.2 ± 1.3 38.7 ± 2.1 Heart wet weight (mg) 258 ±6  242 ± 14 Heart rate (beats per minute, bpm) 368 ± 23 345 ± 23Developed force (g)  0.81 ± 0.19  1.12 ± 0.12 Rate force product (g/bpm)308 ± 80 372 ± 49 Coronary flow (ml min 1)  2.3 ± 0.2  1.7 ± 0.1Values are the mean±standard error of the mean (SEM). No significantdifference (p>0.05) between the groups was found for the listedparameters, except coronary flow.

Component Retention Times, Inosine Calibration and AUC Calculations

During HPLC method development and validation, combined standards ofadenosine, inosine, hypoxanthine, xanthine and uric acid were preparedin Krebs buffer solution at concentration levels of 1, 2.5, 5, 10 and 25μg ml⁻¹. Standard curve linearity (non-weighted) of all components wasacceptable with all correlation coefficients >0.995. During subsequentanalytical runs, a single point calibration standard mixture containing2.5 μg ml⁻¹ of each component was prepared in Krebs buffer solution andwas used to identify component retention times and the quantification ofinosine found in test samples. Using ultraviolet light detection at 240nm, component peak area and external standardization were used forinosine computations. To determine inosine AUC on the test samples, thetrapezoidal rule computation using Excel® software (a spread sheetavailable from Microsoft Corporation) was performed on inosine samplevalues from 0 to 20 min.

Results and Discussion Initial Evaluation for (.OH) Free Radicals

During periods of cardiac oxidative stress (e.g. acute myocardialinfarction), the heart is deprived of the oxygen needed for ATPsynthesis. In the absence of oxygen, dormant enzymes activate wherebyATP is sequentially converted to ADP, AMP, adenosine, inosine andhypoxanthine. Upon reperfusion of the heart with oxygenated blood oroxygenated Krebs solution, additional cellular enzymatic conversionstranspire with the xanthine oxidase converting hypoxanthine to xanthineand uric acid. A metabolic by-product of xanthine oxidase is theformation of hydrogen peroxide (H₂O₂), which is normally converted byglutathione peroxidase to H₂O. However, in the presence of Fe₂, H₂O₂ maybe converted to a hydroxyl free radical (.OH) via the Fenton and HaberWeiss reactions (FIG. 1) (IUPAC 1997).

The (.OH) is a known potent reactive oxygen species (ROS) and can causedamage to cellular components (e.g. lipids, proteins, nucleic acids)(Tardif & Bourassa 2000). To investigate the formation of ROS, oneresearch objective was to evaluate and estimate the amount of (.OH)generated from 20 min of global cardiac ischemia using isolated mousehearts. In several of the initial experiments, SA (1 mM) was fortifiedin the Krebs buffer solution (pH adjusted 7.4) to react with (.OH) andform the reaction products of 2,3- and 2,5-DHBA isomers (Onodera &Ashraf 1991, Coudray & Favier 2000). The HPLC-DAD conditions that wereused for inosine determination resolved prepared standards (13 ng ml 1or 86 nM) of the 2,3- and 2,5-DHBA isomers from other Krebs eluantsample components (e.g. SA, adenosine, inosine, hypoxanthine, etc.).

However, in the experiments performed using SA we did not observe eitherthe 2,3- or 2,5-DHBA isomers in the sample chromatograms from mousehearts subjected to global cardiac ischemia. It is possible that theinitial level of SA (1 mM) added to the Krebs buffer solution increasedthe total solute concentration to a level, which reduced the solubilityof 2,3- and 2,5-DHBA isomers and therefore made each analyticallyundetectable. Lower concentrations of SA (e.g. ≦1 μM) may in theoryresolve this aspect of ROS generation from mouse global cardiacischemia.

HPLC-DAD and HPLC-MS Confirmation

The HPLC-DAD method was used for determining all of the followingcomponents (adenosine, inosine, hypoxanthine, xanthine, uric acid, 2,3-and 2,5-DHBA isomers). The mobile phase aqueous component 0.05% TFA indeionized water was chosen as a pH of approximately 2.3 provided goodpeak shapes on all components and a low pH was necessary to reduce peaktailing on the acidic components (e.g. 2,5-DHBA has a pKa ˜2.9). TheSynergi™ Hydro-RP C₁₈ (polar endcapped) and Synergi™ Polar-RP C₁₈(ether-linked phenyl) columns of identical dimensions were evaluated foruse. While both columns worked well for inosine and polar components(e.g. adenosine), the Synergi™ Hydro-RP C₁₈ was selected for overallanalysis as it provided good component peak shape and sufficientresolution of all components.

Other components evaluated using this method have HPLC retention timesas follows: uric acid, 2.8 min; hypoxanthine, 3.9 min; xanthine, 4.2min; adenosine, 5.7 min; CK-MB, 8.2 min; 2,3-DHBA, 8.4 min; 2,5-DHBA,10.2 min; myoglobin, 14.1 min; atrial natriuretic peptide, 14.5 min;brain natriuretic peptide, 15.0 min; and salicylic acid, 15.4 min. Bothtroponin I and troponin T were not detected using this HPLC method. AnHPLC-DAD chromatogram overlay from a mouse subjected to 20-min globalcardiac ischemia and a control mouse (non-ischemia) are presented inFIG. 2 with inosine elution at 5.9 min.

To evaluate perfusate sample stability, the prepared samples wereinitially injected and analysed by HPLC-DAD. The samples weresubsequently stored overnight on the autosampler at ambient laboratorytemperature and re-injected (n=3 times) to evaluate both for changes incomponent levels due to possible synthesis or degradation reactions frompotential enzymes eluted in the perfusate and to evaluate instrumentprecision. In all re-injected perfusate samples, component levelsremained constant (5/4% RSD) indicating stability overnight at ambienttemperature and the absence of appreciable levels of nucleoside andpurine converting enzymes in the perfusate.

HPLC-MS Confirmation of Inosine as Potential Initial Ischemia Biomarker

An HPLC-MS was used to confirm inosine at retention time 5.9 min insamples from test mice subjected to oxidative stress. The HPLCanalytical column, mobile phase gradient and flow rate were identical tothat used in the HPLC-DAD method. The mass spectrum for inosine (MW=268Da) is presented in FIG. 2. It was acquired using the MS positive-ionmode, which provided a good mass spectral quality match against aprepared standard of inosine in Krebs buffer solution. The full-scanspectrum was achieved using up-front collision-induced dissociation(CID) and nitrogen as the collision gas. The mass spectrum base peak(137 Da) represents the cleavage of the ribose entity from inosineleaving a protonated hypoxanthine (MW=136 Da).

Evaluation of Inosine AUC and Other Cardiovascular Parameters

Initially, the focus was on identifying cardiac protein or peptidebiomarkers (e.g. Atrial Natriuretic Peptide, Brain Natriuretic Peptide,that may be released from ischemic myocardium; however, in comparisonwith non-ischemic mouse hearts only inosine (22-69-fold) andxanthine-like products (e.g. hypoxanthine (>7×), xanthine (approximately3×), uric acid (approximately 3×) were found at higher levels inglobally ischemic mouse hearts. FIG. 3 is a representative profile ofone mouse heart subjected to oxidative stress with the individual ATPdegradation by-product components DAD response plotted against Krebsbuffer reperfusion time. As can be seen, inosine was the component thathad the highest DAD response with detectable component amounts found atlower mg ml 1 levels (e.g. in a range of 0.4-7.5 μg ml⁻¹ in mouse #874)in the sample less than 5 min following reperfusion.

Other cardiovascular parameters (e.g. the percent cardiac ventricularfunctional recovery rate) were measured and reported with the calculatedinosine AUC results (Table 2). As can be seen in Table 2, inosine effluxwas present in test mouse heart perfusate samples that were subjected tooxidative stress and was not detected in control mouse heart perfusatesamples. However, for both controls and test mice, the percent cardiacfunctional recovery rate ranged from 39 to 92%, with the lowest measuredcardiac functional recovery being in test mouse hearts that had thelargest amount of inosine present in the Krebs buffer solution (e.g.test mouse with 2469 ng min ml⁻¹ AUC inosine effluxed with a 39% cardiacfunctional recovery rate). This may indicate that mouse hearts areinjured to a greater degree from the effects of oxidative stress effluxmore inosine from ATP by-product degradation.

TABLE 2 Inosine washout and cardiac ventricular functional recovery inLangendorff mouse hearts following aerobic perfusion and 20-min globalischemia. Inosine area under the curve (AUC) 0-20 min Cardiac FunctionalSample type (ng min ml⁻¹) recovery rate (%) Control n.d.* 70 Controln.d. 72 Control n.d. 74 Control n.d. 82 Control n.d. 81 Control n.d. 64Test 653 92 Test 962 84 Test 954 77 Test 1003 53 Test 2469 39 Test 258352 *n.d., Not detected.

Conclusions

These results suggest that the level of inosine found in test animalssubjected to cardiac oxidative stress may serve as a biomarkerindicative of early cardiac ischemia. This can be explained by ischemicmyocytes undergoing nucleotide purine catabolism in the absence ofoxygen with subsequent activation of dormant cellular enzymes and thegeneration of degradative breakdown products of ATP.

References for Example 1

-   ADAM, Inc. 2005. Heart attack and acute coronary syndrome (available    at the website located at adam.about.com/reports/-   Abd-Elfattah A S, Higgins R S D, Latifi R, Merrell R C. 2001.    Targeting post-ischemic reperfusion injury: scientific dream and    clinical reality. New Surgery 1:41 51.-   Beyerle K. 2002. POC testing of cardiac markers enhances ED care.    Nursing Management 33(9):37 39.-   Bhagavan N V, Lai E M, Rios P A, Yang J, Ortega-Lopez A M, Shinoda    H, Honda S A A, Rios C N, Sugiyama C E, Ha C. 2003. Evaluation of    human serum albumin cobalt binding assay for the assessment of    myocardial ischemia and myocardial infarction. Clinical Chemistry    49(4):581 585.-   Coudray C, Favier A. 2000. Determination of salicylate hydroxylation    products as an in-vivo oxidative stress marker. Free Radical Biology    and Medicine 29(11):1064 1070.-   Dohm M. 2004. Origin and maintenance of the Hsd:ICR random-bred    strain (available at website located at    www2.hawaii.edu/approximately dohm/Phd/OriginHsd.htm)-   Dorner T, Rieder A. 2004. Risk management of coronary heart    disease-prevention. Wiener Medizinische Wochenschrift 154(11 12):257    265.-   IUPAC. 1997, Fenton and Haber Weiss reactions. IUPAC Compendium of    Chemical Terminology 69: 1274 1277.-   Lees K. 2000. Multiple marker test quickly identifies high-risk    heart attack patients, study says. Heart Signs, Duke University News    and Communications available at website located at dukenews.duke.edu-   Luo J, Jankowski V, Gungar N, Neumann J, Schmitz W, Zidek W,    Schluter H, Jankowski J. 2004. Endogenous diadenosine    tetraphosphate, diadenosine pentaphosphate, and diadenosine    hexaphosphate in human myocardial tissue. Hypertension 43(5):1055    1059.-   Mei D A, Gross G J, Nithipatikom K. 1996. Simultaneous determination    of adenosine, inosine, hypoxanthine, xanthine, and uric acid in    microdialysis samples using microprobe column high-performance    liquid chromatography with a diode array detector. Analytical    Biochemistry 238:34 39.-   Morrow D A, De Lemos J A, Sabatine M S, Antman E M. 2003. The search    for a biomarker of cardiac ischemia. Clinical Chemistry 49(4):537    539.-   Naudziunas A, Jankauskiene L, Kalinauskiene E, Pilvinis V. 2005.    Implementation of the patient education about cardiovascular risk    factors into a daily routine of the Cardiology Unit of the hospital.    Preventive Medicine 41(2):570 574.-   Nelson D, Cox M. 2000. Lehninger principles of biochemistry. 3rd ed.    New York, N.Y.: Worth. p. 848 868.-   Okrainec K, Banerjee D K, Eisenburg M J. 2004. Coronary artery    disease in the developing world. American Heart Journal 148(1):7 15.-   Onodera T, Ashraf M. 1991. Detection of hydroxyl radicals in the    post-ischemic reperfused heart using salicylate as a trapping agent.    Journal Molecular Cellular Cardiology 23:365 370.-   Tardif J, Bourassa M. 2000. Antioxidants and cardiovascular disease.    Dordrecht: Kluwer. p. 57 70.-   Viegas T X, Omura G A, Stoltz R R, Kisick J. 2000. Pharmacokinetics    and pharmacodynamics of peldesine (BCX-34), a purine nucleoside    phosphorylase inhibitor, following single and multiple oral doses in    healthy volunteers. Journal Clinical Pharmacology 40:410 420.-   Xi L, Hess M L, Kukreja R C. 1998. Ischemic preconditioning in    isolated perfused mouse heart: Reduction in infarct size without    improvement of post-ischemic ventricular function. Molecular and    Cellular Biochemistry 186:69 77.

Example 2 An HPLC Method for Determination of Inosine and Hypoxanthinein Human Plasma from Healthy Volunteers and Patients Presenting withPotential Acute Cardiac Ischemia

A simple and sensitive high-performance liquid chromatography (HPLC)method utilizing ultraviolet (UV) detection was developed for thedetermination of inosine and hypoxanthine in human plasma. For componentseparation, a monolithic C18 column at a flow rate of 1.0 mL/min with anaqueous mobile phase of trifluoroacetic acid (0.1% TFA in deionizedwater pH 2.2, v/v) and methanol gradient was used. The method employed aone-step sample preparation utilizing centrifugal filtration with highcomponent recoveries (˜98%) from plasma, which eliminated the need of aninternal standard. The method demonstrated excellent linearity (0.25-5g/mL, R>0.9990) for both inosine and hypoxanthine with detection limitsof 100 ng/mL. This simple and cost effective method was utilized toevaluate potential endogenous plasma biomarker(s), which may aidhospital emergency personnel in the early detection of acute cardiacischemia in patients presenting with non-traumatic chest pain.

Introduction

According to a recent report by the World Health Organization (WHO, 2002data), approximately 32 million myocardial infarctions (MI) occurredworldwide resulting in more than 12 million deaths [1]. Cardiovasculardisease is the leading cause of mortality in the world and includes MI,which can be presaged by acute cardiac ischemia [2-5]. In a patientsuspected of having an MI or on-going acute cardiac ischemia, standarddiagnostic procedures include patient history and physical exam, anelectrocardiogram (ECG) and sequential assessment of biomarkers ofmyocardial damage [6-8].

Current test methods for endogenous cardiac biomarkers (e.g. cardiactroponin I, creatine kinase-MB and myoglobin) include LC-MSanalysis[9,10] and fluorescence immunoassay [11-14]; however elevation of theseprotein biomarkers reflect some level of myocardial necrosis, and aretypically elevated in a diagnostic range several hours after acutemyocardial infarction. Inosine (9-β-d-ribofuranosylhypoxanthine, MW 268Da nucleoside) and hypoxanthine (1,7-dihydro-6H-purin-6-one, MW 136 Dapurine) are endogenous non-protein plasma constituents normally found atlow concentrations (e.g. ˜200-400 ng/mL) in human plasma resulting fromdietary and endogenous purine metabolism [15]. As described in Example1, inosine levels increase from cardiac tissue subjected to constantconditions of oxidative stress (e.g. acute cardiac ischemia ormyocardial infarction).

Current methods for plasma level measurement of selected ATP catabolicby-products such as inosine, hypoxanthine, xanthine and uric acid, inplasma utilize HPLC-UV with sample preparation steps including solidphase extraction [15], protein precipitations (e.g. ethanol or TCA) aswell as some methods requiring use of an internal standard [17,18]. Highperformance liquid chromatography (HPLC) with ion pairing reagents[19-21] or protein precipitation and enzyme catalyzed luminescencedetection [22] have also been used. One HPLC method utilized centrifugalfiltration for sample preparation; however this method did notcompletely resolve hypoxanthine and xanthine components and columndegradation was reported after 3 months of use [23]. None of thesetechniques, however, offers as simple a determination for inosine andhypoxanthine (can also evaluate uric acid, adenosine and xanthine) inhuman plasma as the method described herein. The method utilizescentrifugal membrane filter technology and does not require the use ofan internal standard. In addition, this method employs a recentlyintroduced HPLC column technology (Onyx™ monolithic column, Phenomenex®Inc. 2005 market introduction) [24], which provides sufficient componentresolution and sensitivity for measurement of inosine and hypoxanthinein human plasma samples, from healthy volunteers and emergency roompatients presenting with chest pain with and without acute cardiacischemia.

Experimental Chemicals and Blank Plasma

Hypoxanthine and xanthine were purchased from Acros Organics (Fair Lawn,N.J., USA) and adenosine, inosine and uric acid were purchased fromSigma-Aldrich (St. Louis, Mo., USA) with all chemicals being ACS reagentgrade or higher purity. For mobile phase preparation, trifluoroaceticacid (TFA) was reagent grade, methanol was Optima HPLC grade and bothwere purchased from Fisher Scientific (Fair Lawn, N.J., USA). Ultrapuredistilled and deionized water (18M cm) used for all HPLC work wasprepared in-house using PureLab®Ultra water purification system (USFilter, Lowell, Mass., USA) and 0.2 m filtered prior to use. Blood bankhuman blank plasma used for preparation of controls was provided by VCUMedical Center, Richmond, Va., USA.

HPLC Equipment and Mobile Phase

The HPLC-DAD (diode array detector) equipment consisted of aHewlett-Packard (HP) Model 1090 HPLC system (Agilent Technologies, PaloAlto, Calif., USA). The analytical column used was a Phenomenex® Onyx™monolithic C₁₈, 20 cm×4.6 mm I.D., 130 Å column coupled to an Onyx™ C₁₈guard column, 5 cm×4.6 mm I.D. (Torrance Calif., USA). The guard columnwas replaced after each analytical run of approximately 50 samples. Themobile phase consisted of aqueous trifluoroacetic acid (0.1% TFA indeionized water, pH 2.2, v/v) and methanol gradient. The mobile phasegradient was programmed with time course as follows (99:1 0.1% TFA indeionized water:methanol (v/v) at 0 min and held for 3 min; 70:20 0.1%TFA in deionized water:methanol (v/v) at 10 min; 5:90 0.1% TFA indeionized water:methanol (v/v) at 11 min and held 2 min, and 99:1 0.1%TFA in deionized water:methanol (v/v) at 14 min). The mobile phase wascontinuously degassed using helium sparging and used at a flow rate of1.0 mL/min. Typical HPLC operating pressure at gradient time 0 minconditions was approximately 84 bar with ambient column temperature. Aninjection volume of 15 L of the prepared plasma sample was accomplishedusing the HP Model 1090 autosampler. Component detection was achievedusing the HP Model 1090 DAD detector with data collection at the optimalUV wavelength absorption of 250 nm for both inosine and hypoxanthine.The detector was operated at high sensitivity set point with a 1 sresponse time. A 345 kPa backpressure regulator (SSI, State College,Pa., USA) was coupled to the detector outlet to prevent mobile phaseoutgassing. Data acquisition and component computations were performedusing TotalChrom™ Workstation software (Perkin Elmer™, Norwalk, Conn.,USA).

Standard and Control Preparation

Stock standards of adenosine, inosine, hypoxanthine, xanthine and uricacid (100 g/mL) were prepared in deionized water and stored at 4° C.Working standards to establish HPLC retention times of adenosine,xanthine and uric acid components were prepared at 2.5 μg/mLconcentrations in deionized water. Working standards of inosine andhypoxanthine (250, 500, 1000, 3000 and 5000 ng/mL) were prepared indeionized water. All working standards were stored at −70° C. and stablefor at least 6 months. Working controls of inosine and hypoxanthine(250, 2000 and 4000 ng/mL) were prepared using pooled hospital bloodplasma (n=3 donated lots) which were evaluated individually andconfirmed to lack detectable levels of inosine and hypoxanthinecomponents.

It is possible the levels of inosine and hypoxanthine in blood bankplasma were not detectable due to the time (>10 days) the plasma wasstored refrigerated (4° C.) prior to expiration and availability forlaboratory experimental use. Without freezing the plasma or utilizingplasma enzyme inhibitors, xanthine oxidase and purine nucleosidephosphorylase found in plasma may metabolize the normally low levels ofinosine and hypoxanthine to their end product uric acid. Followingpreparation of control samples, they were immediately frozen at −70° C.,to prevent endogenous plasma purine nucleoside phosphorylase fromconverting inosine to hypoxanthine prior to formal sample analysis.Following hospital approval, blood was obtained from hospital emergencyroom patients (n=20), in vacutainer TM tubes containing heparin as perhospital emergency room protocols for patients presenting with chestpain and potential MI or acute myocardial ischemia. Sample tubes werecentrifuged at ˜1000×g for 10 min with plasma drawn off and split intotubes for hospital clinical testing and one tube immediately frozen at−20° C. (transferred to −70° C. for storage) for inosine andhypoxanthine analysis. Plasma samples from healthy blood donors (maleand female, both genders >18 years of age) were purchased from ProMedDx(Norton, Mass., USA) which used an IRB approved specimen collectionprotocol and stored frozen at −70° C. Prior to HPLC analysis, plasmasamples were thawed to ambient temperature, mixed thoroughly byinversion and centrifuged at 1000×g for 10 min to eliminate fibrinousmaterial.

Sample Preparation

Samples were prepared for HPLC analysis by pipetting 250 μL of plasmainto a polypropylene Microcon® YM-10 (10,000 molecular weight cutoff,MWCO) centrifugal filter tube (Millipore, Bedford Mass., USA). Thesample tubes were capped and centrifuged at 14,000×g for 15 min atambient lab temperature. The clear filtrates were transferred todeactivated glass HPLC autosampler vials (Waters®, Milford Mass., USA)with 15 μL injected into the HPLC system for analysis.

Results and Discussion HPLC Conditions Optimization

Several types of C₁₈ columns were evaluated for resolving adenosine,inosine, hypoxanthine, xanthine and uric acid from other plasmacomponents. Due to minimal sample preparation using the centrifugalmembrane filter, the ideal HPLC column should have high efficiency forresolving inosine and hypoxanthine components from components in theplasma matrix. Conventional HPLC columns such as Synergi Polar-RP C₁₈(15 cm×3.0 mm I.D.×4 μm packing) and Hypersil ODS C₁₈ (15 cm×3.2 mmI.D.×3 μm packing) were evaluated versus the recently marketed HPLCcolumn technology, the Onyx monolithic C₁₈ column (10 cm×4.6 mm I.D.).The monolithic column provided superior chromatographic resolution ofcomponents with a low system backpressure of approximately 84 bar(gradient time zero conditions and flow rate of 1 mL/min). It should beemphasized that both conventional HPLC columns were evaluated atoperating flow rates of ˜0.6 mL/min and with system pressures that wereapproximately twice as high as when using the monolithic column. Thesupplier of the monolithic column cited advantages of high componentefficiencies (resolution) and low system backpressure with use of thenew monolithic column technology. We observed that both of these statedadvantages over the two conventional mid-bore diameter HPLC columnsevaluated were clearly demonstrated.

The mobile phase aqueous component, 0.1% TFA in deionized water,provided a pH of 2.2 which also provided good peak shape (e.g. uric acidcomponent, pKa ˜5.8) from components of interest from the endogenousplasma components (MW<10,000 Da) obtained from the YM-10 samplepreparation. Optimization and adjustment of the acid strength improvedthe separation between hypoxanthine (RT 5.2 min) and uric acid (RT 5.7min). Initial use of aqueous 0.05% TFA did not provide componentbaseline resolution while aqueous 0.1% TFA offered complete componentbaseline resolution at the expense of increased column retention times.The mobile phase organic modifiers (e.g. acetonitrile versus methanol)were evaluated to determine which organic solvent would provide the bestchromatographic separation from endogenous plasma components and at thesame time being most cost effective. Methanol was chosen as the organicmodifier as it provided symmetrical component peak shapes and goodselectivity from other endogenous plasma components; however the HPLCsystem backpressure was somewhat higher when using methanol with themethanol gradient increasing from 1 to 90%. Methanol is also more costeffective for routine HPLC analysis because of its lower procurementcost.

A mobile phase gradient was used for reproducible separations of thestructurally similar purines (hypoxanthine, uric acid) and nucleosides(inosine, adenosine). Since the mobile phase organic constituent iscritical to controlling component elution times (initial 1% methanolcomposition at gradient time zero), the use of protein precipitationtechnique using solvents such as acetonitrile or methanol (typically 1:1or 2:1, organic:plasma ratio) was eliminated from consideration. Thestructurally similar components injected using organic solventprecipitation were not chromatographically resolved due to bandbroadening effects from the added organic modifier. Different columnoven temperatures (e.g. ambient lab of 20, 30 and 40° C.) were evaluatedwithout significant chromatographic improvement (component resolution,peak shape), thus ambient temperature was utilized for the analysis. Athigher column temperatures (e.g. 40° C.), component co-elution for bothearly (hypoxanthine, uric acid) and late components (inosine, adenosine)was observed.

Linearity, Limits of Quantitation and Detection, Computations

The plasma method was linear throughout the concentration range of0.25-5 μg/mL for inosine (mean correlation coefficient of 0.9991, n=10)and hypoxanthine (mean correlation coefficient of 0.9998, n=10) with allstandard back-calculated values within 5% of their nominal amount. Thelimit of detection (LOD) for each component of the method was ˜100ng/mL. The LOD was determined using a fortified amount of each componentin pooled blood blank plasma at 100 ng/mL (n=3) and calculation fromeach component's standard curve (component peak heights had greater thanthree times s/n than blank plasma background). For plasma componentcalculations and reporting results, normal linear regression utilizingexternal standardization and peak height was used with the loweststandard calibrator (0.25 μg/mL) used as the limit of quantitation(defined as combined accuracy and precision within 20% of the nominalamount).

Accuracy, Precision and Recovery

The accuracy and precision for the method was determined by evaluationof replicate prepared plasma control samples at 250, 2000 and 4000 ng/mL(Table 3). The combined intra-day (within day) and inter-day (betweenday) accuracy of the method was reported as the percent error of nominalfortified amounts versus measured component concentrations. The combinedintra-day and inter-day precision of the method was reported as percentrelative standard deviation (% R.S.D.). The method demonstratedexcellent accuracy (±6%) and precision (±8.1) for both components inplasma (n=15 at each component concentration level).

TABLE 3 Combined intra- and inter-day accuracy and precision for inosineand hypoxanthine in plasma controls Fortified concentration Calculatedmean (ng/mL) concentration Error R.S.D. Component (n = 15) (ng/mL) (n =15) (%) (%) Inosine 250 243 −2.8 8.1 Inosine 2000 1966 −1.7 4.9 Inosine4000 3914 −2.2 3.6 Hypoxanthine 250 265 6.0 7.5 Hypoxanthine 2000 20442.2 5.5 Hypoxanthine 4000 3931 −1.7 2.2Controls demonstrated excellent accuracy±6% and precision±8.1%throughout the plasma concentration range.

Absolute recovery for the plasma method was evaluated by comparingextracted fortified controls prepared in pooled blood blank plasmaversus unextracted standards prepared in deionized water (n=3 at 250,2000 and 4000 ng/mL). The absolute recovery for the plasma method wasdetermined to be >98% for both inosine and hypoxanthine. In addition,the standards and controls used for all HPLC analysis were prepared andhandled identical to patient and volunteer subject samples, thuscontrolling for potential errors in sample handling, micropipetting andYM-10 component extraction recovery.

Chromatography

FIGS. 4 A-E illustrate chromatograms of 2000 ng/mL hypoxanthine (RT ˜5.3min), uric acid (RT ˜5.8 min), xanthine (RT ˜7.2 min), adenosine (RT˜10.7 min) and inosine (RT ˜10.9 min) in deionized water for markingcomponent retention times; limit of quantitation and lowest plasmastandard of 250 ng/mL hypoxanthine and inosine; pooled blank plasma fromthe VCU Health Systems Hospital blood bank; prepared plasma from ahealthy female subject; and prepared plasma from a hospital emergencyroom female patient exhibiting symptoms of chest pain and acutemyocardial ischemia (FIGS. 4 A, B, C, D and E, respectively). The methoddemonstrated excellent chromatographic selectivity with no endogenousplasma interferences at the retention times of hypoxanthine and inosinewith sufficient sensitivity for both components of interest usingconventional UV detection and an analytical run time of ˜21 min (allowsmobile phase gradient equilibration). To extend column lifetime, theanalytical column was flushed after each analytical run (˜50 injections)for 1 h at 1.0 mL/min with acetonitrile: deionized water (90:10, v/v) toeliminate potential retained nonpolar substances from the column.

Sample Preparation, Optimization and Filtrate Stability

Sample preparation evaluations using protein precipitation andcentrifugal membrane filters were conducted. As previously described,organic solvent precipitation was not useful due to resulting poorchromatographic resolution of structurally similar components. TCA wasnot evaluated due to the hazards of using the strong acid and theresulting sample dilution effect potentially affecting overall methodsensitivity. The centrifugal membrane filter is commonly used toconcentrate peptides, proteins and nucleic acids for proteomic andgenomic determinations [25]. Since the molecular weights of ourcomponents are all less than 300 Da, our approach to using thistechnique was to inject the filtrate which would contain the lowmolecular weight components that transfers across the YM-3 or YM-10cellulose membrane cutoff filters. This essentially removes mostpeptides and all proteins from the sample to be injected as they areretained by the cellulose membrane cutoff filter, thus improving methodselectivity. Method sensitivity is also improved because there is nosample dilution effect since no solvent is added.

Evaluations to optimize sample preparation conditions using the YM-10(10,000 Da MWCO) and YM-3 (3000 Da MWCO) centrifugal filter wereconducted. With the centrifugal force set at 14,000×g (recommended byYM-10 supplier) and using 250 μL of prepared plasma control samples, thecentrifuge spin time was varied from 5, 15, 30 and 60 min. The 5 minspin time did not provide enough time to adequately separate plasmaproteins from the aqueous matrix (salts, small peptides and substancesless than 10,000 Da) with an insufficient amount of sample filtraterecovered. The 15, 30 and 60 min centrifugal spin times resulted inmaximum recovery of sample filtrate. However, the 60 min spin filtratesamples were significantly warmer than ambient lab temperature mostlikely due to warming effects of the sample tubes friction with air fromthe centrifugal spin. Thus to eliminate potential component degradationdue to heat from spinning 60 min and to shorten sample preparation time,a spin time of 15 min was used for all analyses as above.

Results for the YM-3 filter evaluation demonstrated longer spin timeswere required (˜45-60 min) at 14,000×g versus the 15 min spin using theYM-10 filter. The YM-3 filtrate did not offer better filtration ofsmaller plasma peptides (<10K Da), as observed on chromatograms, thanwas already achieved using the YM-10 filter. However, using either YM-3or YM-10 filter effectively removed the purine nucleoside phosphorylaseenzyme (nominal weight ˜90-94 kDa protein, [26,27]) thus eliminating thepotential for inosine to hypoxanthine metabolism in the sample filtrate.The filtrates were stored frozen (−70° C.) after HPLC analysis with bothinosine and hypoxanthine components demonstrating stability for greaterthan 3 months.

Plasma Purine Nucleoside Phosphorylase Activity

Purine nucleoside phosphorylase (PNP, EC 2.4.2.1) is an enzyme thatrapidly metabolizes inosine to hypoxanthine in blood (t½<5 min due tored blood cells). This enzyme has low activity in plasma and is normallyfound in human cardiac muscle, GI tract, spleen, brain and red bloodcells [28,29]. Therefore, to better estimate an ischemic heart'seffluxed inosine during periods of acute cardiac oxidative stress,venous blood samples should be kept cold (ice) and prepared immediately.Either the blood sample should be immediately inhibited (e.g. peldesine,competitive inhibitor [28]) or the metabolite hypoxanthine should besimultaneously determined with inosine to better estimate the level ofacute cardiac ischemia. In whole blood or plasma samples, hypoxanthinewill not be further metabolized to xanthine as the human enzyme xanthineoxidase (XO), which is required for hypoxanthine to xanthine conversion,has low activity in plasma [30] and being typically found in humantissue (liver, small intestine) and other bodily fluids (milk,colostrum). A plasma (heparinized) sample is recommended for inosine andhypoxanthine determination in that the approximate 30 min clot timerequired for a serum sample would allow significant conversion ofinosine to hypoxanthine in the collection tube, which

would contain PNP from the red blood cell and plasma matrix.

Several evaluations (n=3 samples at each condition) were performed toevaluate inosine metabolism by PNP activity in plasma stored at 4° C.(refrigerator), −20 and −70° C. Results of the 4° C. evaluation can beseen in FIG. 5; plasma fortified with inosine only at 2000 ng/mL andwithout PNP enzyme inhibitor is metabolized rapidly to hypoxanthine(˜70% in 24 h); plasma fortified with 250 ng/mL of inosine andhypoxanthine and without PNP enzyme inhibitor is also metabolizedrapidly to hypoxanthine (˜70% in 24 h); however the plasma fortifiedwith 2000 ng/mL of inosine and hypoxanthine and without a PNP enzymeinhibitor, is metabolized less rapidly to hypoxanthine (˜30% in 24 h)and slightly less than 50% after 72 h. Results of storing fortifiedplasma samples at −20° C. immediately after preparation indicated areduced rate of inosine to hypoxanthine conversion (˜30% after 8 months)with storage at −70° C. almost completely deactivating the PNP enzyme(<5% inosine conversion after 3 months).

A possible explanation for the plasma hypoxanthine concentrationdependence for the conversion rate of inosine to hypoxanthine would beproduct inhibition (PNP K_(eq) ˜0.04 mM) [31]. This low Keq indicatesthat thermodynamically, inosine synthesis is favored over productconversion to hypoxanthine. When the venous sample plasma concentrationof hypoxanthine is present at higher levels (e.g. 2000 ng/mL), theconversion of inosine to hypoxanthine by plasma PNP decreases in theabsence of significantXOenzyme activity, which converts hypoxanthine toxanthine and uric acid for biological elimination (therefore XO activityultimately increases PNP activity as it reduces hypoxanthine productinhibition of PNP). It was also determined that the total amount ofinosine and hypoxanthine fortified into the pooled plasma was recovered,thus verifying the lack of significantXOactivity in human plasma andsupports our recommendation of simultaneous determination of bothinosine and hypoxanthine components. A preliminary investigation to

show the utility of the method is shown in FIG. 1D (healthy control with350 ng/mL inosine and 373 ng/mL hypoxanthine) and 1E (potential acutecardiac ischemia patient with 641 ng/mL inosine and 3987 ng/mLhypoxanthine). These figures demonstrate an increase in both inosine andhypoxanthine concentrations in one patient having presented with chestpain and undergoing evaluation for acute cardiac ischemia.

Conclusions

A sensitive and selective method has been developed for evaluation ofinosine and hypoxanthine in human plasma. The method employed a one-stepsample preparation for plasma (no organic solvents or solid phaseextraction cartridges required) with high analyte recoveries, whicheliminated the need for an internal standard. In addition, this methodutilized recently introduced HPLC monolithic column technology, whichprovided sufficient selectivity and sensitivity for measurement of thesecomponents. The method was employed without significant methodologicalproblems in the evaluation of plasma samples obtained from healthyvolunteers and hospital emergency room patients presenting with chestpain and potential acute myocardial ischemia.

References for Example 2

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Example 3 Rapid Chemiluminescence Detection of Inosine and HypoxanthineUsing Microplate Luminometer Introduction

This Example describes the development of a rapid chemiluminescence testmethod for determination of inosine and hypoxanthine in human plasma.The purpose is to allow for rapid patient screening capability(diagnostic tool for acute cardiac ischemia) for potential use, forexample, in hospital emergency department environments. The luminescencemethod was tested on samples from healthy individuals and hospitalpatients with confirmed acute MI (hospital documented elevated levels ofcTnT). The method is rapid (defined as less than 10 minute analysistime), sensitive and specific for inosine and hypoxanthine therebyreducing potential errors in interpreting sample test results (e.g.false positive and false negative results are minimized). Currently,there are no rapid test methods to determine inosine and hypoxanthine inplasma, which can meet the stringent sample turnaround time requirementsof an emergency medical services (EMS) environment. The rationale forusing chemiluminescence technology over commonly used liquidchromatography (LC) and immunoassay technologies are as follows: LC andimmunoassay methods are both very sensitive and specific techniques(e.g. monoclonal antibodies for immunoassay and mass spectrometerdetection for LC); however, an LC-MS system is expensive to purchase andoperate, both techniques require technical expertise to perform, andboth lack the rapid turnaround time needed by an EMS facility analyzinga priority “stat” type samples. However, a luminometer can measurechemiluminescent light, is relatively inexpensive to purchase, currentlyused in clinical labs (microplate capability), and can provide highcomponent sensitivity.

Luminescence technology is well established with many instrument vendors(e.g. BMG LabTech Inc. Lumistar Optima (Durham, N.C., USA), BioTekSynergy HT (Winooski, Vt., USA), Thermo Fisher Scientific Luminoskan(Waltham, Mass., USA)) and suppliers (e.g. Corning Life Sciences,Lowell, Mass., USA) of luminescence supplies and reagents availableworldwide. It is known to be one of the most sensitive techniques, withone recent publication, for example, on its application for low ng/mlconcentrations of ATP in human plasma [Gorman et al., 2007]. The highsensitivity of luminescence is primarily due to its high analyte signalto noise (s/n) ratio, with reported detection levels at low picogram andfemtogram levels.

To address biomarker specificity requirement, the subject luminescencetest method will utilize biological enzymes purine nucleosidephosphorylase (PNP) and xanthine oxidase (XO), which are specific forenzymatic conversions of inosine and hypoxanthine, respectively. The PNPenzyme converts inosine to hypoxanthine and XO converts hypoxanthine toxanthine, followed by XO conversion of xanthine to final product uricacid (in human species). Each time XO reacts with one mole ofhypoxanthine, and subsequently with one mole of xanthine, the metabolicby-products of each XO enzymatic turnover is the production of one moleof hydrogen peroxide and two moles of superoxide anion radical (O²—).Both of these by-products can become substrates for luminescence typereactions. Several commonly used luminescent materials (e.g. luminol(oxidation), lucigenin (reduction), and PHOLASIN® (oxidation) wereconsidered for this research. If using luminol or lucigenin as theluminescent material, the hydrogen peroxide (which has both oxidizingand reducing capabilities) can react with the horseradish peroxidase(HRP) enzyme, luminol, and signal enhancers to generate measurable bluelight ˜450 nm, thus an amplification of signal effect (one mole ofhypoxanthine and xanthine can generate two moles of hydrogen peroxide)(FIG. 6).

However, to achieve even greater sensitivity at low concentrations(ng/ml or μM levels of inosine and hypoxanthine are typically found inhuman plasma) another luminescence approach was investigated, whichutilizes a highly sensitive photoprotein (PHOLASIN®). Since one mole ofhypoxanthine will generate 4 moles of superoxide anion radicals (SAR) asa by-product of XO activity, using a chemiluminescent material thatreacts with SAR should theoretically provide even more luminescencesignal, thus potentially increasing the sensitivity two fold over usingthe hydrogen peroxide/horseradish peroxidase/luminol approach. Onearticle cited PHOLASIN® having more than 100 fold sensitivity thanlucigenin (Knight, 1997).

PHOLASIN®, a photoprotein isolated from the bi-valve mollusk, has beenreported to be a very sensitive chemiluminescent material (calledLucidalin®) for SAR and other reactive oxygen species (ROS) such as thehydroxyl free radical (Knight, 1988). PHOLASIN® has been extensivelystudied and patented by Knight Scientific, Plymouth, UK. It is anapproximately 34-36 kDa glycoprotein, which can be made excitable byseveral ROS, emitting blue-green light, and it has been reported to nothave fluorescent properties. The presence of SAR can react with thePHOLASIN® photoprotein to generate measurable light (˜490 nm) (FIG. 7),thus an amplification of signal effect (one mole of hypoxanthine cangenerate four moles of SAR), which should increase sensitivity andprovide lower component detection limits. The reaction of PHOLASIN® withSAR can be very quick (flash type technique, typically seconds) and maybe made even more sensitive with use of signal enhancers (e.g.Adjuvant-k (proprietary) from Knight Scientific).

The Lumistar Optima Microplate Reader (BMG LabTech, Durham, N.C., USA)was used for all luminescence evaluations. The instrument hastemperature control, supports the use of 96 well plates (opaque white)which were purchased from Corning Life Sciences (Lowell, Mass., USA),and is capable of variable microplate mixing speeds with flash and glowluminescence capabilities. The instrument is fitted with two directinjectors capable of rapid injections (e.g. 310 μl/sec), thusmicropipetting assay reagents into the sample wells was automaticallyperformed, which may help to reduce potential errors from manualpipetting.

Experimental Chemicals, Reagents and Materials

Hypoxanthine, xanthine and ethyl alcohol (HPLC grade, denatured) werepurchased from Acros Organics (Fair Lawn, N.J., USA). Inosine, dibasicsodium hydrogen phosphate, and uric acid were purchased fromSigma-Aldrich (St. Louis, Mo., USA). Enzymes xanthine oxidase (isolatedfrom bovine milk, Grade III, ammonium sulfate suspension, enzymaticactivity ˜1.3 units/mg protein, storage temp 2-8° C.), purine nucleosidephosphorylase (isolated from human blood, lyophilized powder, enzymaticactivity ˜19 units/mg protein, storage −20° C.) and uricase (isolatedfrom Arthrobacter globiformis, lyophilized powder, ˜19.7 units/mgprotein, storage −20° C.) were all purchased from Sigma-Aldrich.

A commercial test kit used for antioxidant evaluations was purchased forinitial setup of the luminometer and included an assay utilizingxanthine/xanthine oxidase plate mode kinetics (glow technique). The kitincluded PHOLASIN® (50 μg), xanthine, xanthine oxidase [˜10.25 mU/ml]and buffer (proprietary) for plate mode kinetics and was purchased fromKnight Scientific (Plymouth, UK). The luminometer instrument wasqualified using the commercial antioxidant test kit and by successfulreplication of the xanthine/xanthine oxidase plate mode kinetics profilefrom Knight Scientific. For all experiments following instrumentqualification, the reagents and enzyme solutions were preparedaccordingly. Dibasic sodium hydrogen phosphate was used to prepare the20 mM assay buffer solution with ultrapure deionized water as thediluent (final pH 7.4 using concentrated phosphoric acid). Ultrapuredeionized water (˜18 MO-cm) used for all reagent solutions was filtered(0.2 μm) prior to use.

The luminometer rinse solution for the direct injector syringes wasprepared using ethyl alcohol:deionized water mixture (75:25%, v/v).Weekly rinses were performed to reduce potential material (e.g. proteinand enzyme residue) buildup in the syringes, reagent tubing and injectorneedles. Opaque 96 well microplates were purchased from Corning LifeSciences (Lowell, Mass., USA) and stored in the dark at ambienttemperature. Blank human plasma (lithium anticoagulant) from one healthyvolunteer (250 ml), an additional six healthy volunteers plasma (lithiumheparin) samples (1 ml each), and six patient's plasma (lithium heparin)samples with confirmed acute MI (hospital reported elevated cTnT, 1 mleach) were purchased from ProMedDx (Norton, Mass., USA) and stored at−20° C. prior to use.

Preparation of Standards, Enzymes and PHOLASIN® Solutions

Stock standards of inosine (25 μg/ml, 93.2 μM), hypoxanthine (25 μg/ml,183.7 μM), xanthine (25 μg/ml, 164.4 μM) and uric acid (25 μg/ml, 148.7μM) were prepared in deionized water, stored at 4° C. with stabilitygreater than 3 months. Working calibration standards for each componentwere prepared in deionized water immediately prior to use. Forexperiments, the working xanthine oxidase solution was prepared bypipetting 40 μL of the aqueous stock XO (from bovine milk) suspensioninto 2.0 ml of assay buffer (pH 7.4) resulting in ˜676 mU XO/ml. Theworking XO solution was stable at ambient laboratory temperature (22°C.) and could be stored at 4° C. overnight with minimal loss in enzymeactivity; however the working XO solution should not be stored frozen(e.g. −20° C.), as a complete loss of enzyme activity was observed uponfreeze-thaw and subsequent use.

To prepare PNP and uricase solutions from solid and lyophilized purinenucleoside phosphorylase and uricase, 1.0 ml of assay buffer (pH 7.4)was pipetted directly into the vendor container bottle with gentlevortexing into solution. After reconstitution using 1 ml of assay buffer(pH 7.4), the PNP stock concentration was ˜18.7 Units PNP/ml and uricasestock concentration was ˜110 Units uricase/ml. A working solution of PNP[˜701 mU PNP/ml] was prepared by pipetting 75 μL of the aqueous stockmaterial into 2.0 ml of assay buffer (pH 7.4). A working solution ofunease [˜1.1 U uricase/ml] was prepared by pipetting 20 μL of theaqueous stock material into 2.0 ml of assay buffer (pH 7.4). Bothworking solutions of PNP and unease were stable at ambient laboratorytemperature and could be stored at 4° C. overnight with minimal loss inenzyme activity.

For preparation of the PHOLASIN® luminescent material, 5.0 ml of assaybuffer (pH 7.4) was pipetted directly into the vendor container bottlecontaining 50 μg PHOLASIN® with gentle vortexing, resulting in a ˜10μg/ml solution. The prepared PHOLASIN® reagent was stable at ambientlaboratory temperature and 4° C., and was stored protected from light toeliminate potential basal luminescence as it is an excitablephotoprotein. The reconstituted PHOLASIN® solution was transferred andstored in plastic screw top tubes (˜1 ml aliquots stored at −20° C.).

Luminometer Equipment and Set Points

The luminometer equipment consisted of a BMG LabTech Inc. LumistarOptima and Optima software (version 2.1) (Durham, N.C., USA) and DellOptiplex 745 PC (Dell, Tex., USA). The luminometer was equipped withtemperature control (8° C. to 45° C.), two direct injectors (minimuminjection volume of 3 μl) with variable injection speeds (10 μ/s to 420μ/s), and microplate shaking (orbital, linear, figure eight) capability.The luminometer listed specifications for the limit of detection (<50amol/well ATP), spectral range (240-740 nm) and dynamic range (9decades). All luminescence assays utilized opaque 96 well plates, anincubation temperature of 25° C., lens mode (no emission filter) and aphoto-multiplier (PMT) gain setting of 3900 volts. Equipment set pointsfor all experiments in the flash mode are listed in FIG. 9.

Method Development and Optimization

Development and optimization of the luminescence test method includedevaluation of parameters such as determining hypoxanthine concentrationlevel range, adjustment of XO enzyme concentration level to reduceanalysis time, and enzyme incubation time (e.g. PNP) to maximizesensitivity and repeatability and to minimize turnaround time (<10 minanalysis). All plasma analysis utilized 20 μl of sample in a finalmicroplate well volume of 200 p. 1 (effectively making a 1:10 dilutionof the plasma sample). Potential endogenous interference (e.g. uricacid) was evaluated to determine quenching effects as this substance hasantioxidant capacity and is typically found in plasma at highconcentrations (e.g. 350-450 μM), especially in gout patients.

The HPLC results from normal volunteers (ProMedDx plasma) andnon-traumatic chest pain patients (Chippenham Hospital ED plasma) wereused to estimate expected plasma concentrations of inosine andhypoxanthine (Table 4). Since the luminometer is a detection device andwill not separate a mixture of components (as does HPLC), it wasnecessary to utilize the PNP enzyme and convert component inosine tohypoxanthine, and then measure the resulting total plasma hypoxanthine(inosine plus hypoxanthine) concentration. Using the XO enzyme,hypoxanthine converts to xanthine, and xanthine to uric acid. Theluminometer measures the light signal generated from the XO reactionwith hypoxanthine and xanthine (XO generates superoxide anion radicalswhich react with the luminescent material PHOLASIN®). Using a μg/ml toμM (micro molar) conversion table (Excel formula computations, Table 5),a standard curve of hypoxanthine was prepared at concentration range of2.3 to 30.3 μM. The initial hypoxanthine concentration range was set tofocus on hypoxanthine concentrations to maximize the luminescence methodsensitivity and detect concentration differences between healthy normalindividuals and non-traumatic chest pain patients (e.g. ˜3 μM for normalindividual and ˜15 μM for lowest observed chest pain patient). Plasmasamples above the highest standard can be diluted with deionized water.The initial range incorporated total inosine and hypoxanthineconcentrations from both healthy normal individuals and non-traumaticchest pain patients (based on n=20 for each group).

TABLE 4 Estimated inosine, hypoxanthine, xanthine and uric acidconcentrations in healthy normal individuals and non-traumatic chestpain patients. Plasma Plasma [ug/mL] [uM] Comments Estimated lowestinosine level 0.10 0.4 Estimated lowest hypoxanthine level 0.10 0.7Assume 100% inosine to 1.1 hypoxanthine conversion Estimated (normals)inosine level 0.30 1.1 Estimated (normals) hypoxanthine 0.30 2.2 levelAssume 100% inosine to 3.3 Estimated levels (normals) from hypoxanthineconversion Feng et al, Ther Drug Mon (2000) 22: 177-183. Estimated(ischemic) inosine level 0.3 1.1 Lowest chest pain patient valueEstimated (ischemic) hypoxanthine 2.0 14.7 level Assume 100% inosine to15.8 Ischemic (based on Chippenham ED hypoxanthine conversion data).Estimated (ischemic) inosine level 7.8 29.1 Highest chest pain patientvalue Estimated (ischemic) hypoxanthine 9.7 71.3 level Assume 100%inosine to 100.3 Ischemic (based on Chippenham ED hypoxanthineconversion data). Estimated (normals) xanthine level 0.9 5.9 Estimatedxanthine levels (normals) from Feng et al, Ther Drug Mon (2000) 22:177-183. Estimated (normals) uric acid level 60.0 356.9 Potential XOinhibitor and luminescence quenching (anti-oxidant). Estimated (normals,high) uric acid 80.0 475.9 Potential XO inhibitor and level luminescencequenching (anti-oxidant). Estimated uric acid highest level (gout) 100.0594.8 Potential XO inhibitor and luminescence quenching (anti-oxidant).

TABLE 5 Component μg/ml to μM conversion table. Compound Weight mgVolume Ml Conc μg/ml Conc μM Adenosine 25.0 1000.0 25.0 93.6 Inosine25.0 1000.0 25.0 93.2 Hypoxanthine 25.0 1000.0 25.0 183.7 Xanthine 25.01000.0 25.0 164.4 Uric Acid 25.0 1000.0 25.0 148.7

Xanthine was found to be at a constant concentration (˜6 μM) in bothnormal individuals and non-traumatic chest pain patient samples. It isimportant to discuss why a standard curve of xanthine would not be usedfor this assay. To prepare a standard curve of xanthine for computationof inosine and hypoxanthine concentrations would report erroneously lowresults, as xanthine only activates the XO enzyme once (xanthine to uricacid), whereas hypoxanthine activates the XO enzyme twice (hypoxanthineto xanthine to uric acid). Since we are only interested in inosine andhypoxanthine concentrations for this research, and with xanthine levelsconstant, it was appropriate to prepare hypoxanthine standards (whichincorporated total inosine to hypoxanthine conversion) for this researchproject.

Typical spreadsheets used for luminescence experiments on inosine,xanthine, and hypoxanthine evaluations include each reagent preparation,volume pipetted into the well, and target concentrations and are listedin Tables 6, 7, and 8, respectively. Using the experimental spreadsheetfor each component standard concentration range, plasma (20 μl) waspipetted into the microplate well with reagents (e.g. assay buffer,phosalin, PNP, uricase) either manually pipetted or injected using onedirect injector; with the other direct injector used to inject the XOsolution to start the reaction with PHOLASIN® and subsequentluminescence emission.

For Table 6: 1) Stock inosine [93.2 μM or 25 μg/ml] in DI and wasprepared by adding 25 mg in 1000 mL DI (or assay buffer); 2) Workingstock (WS) WS-1 (9.32 μM) 100 ul stock inosine (1:10 stock) 900 μl assaybuffer; 3) Final total inosine concentration is based on 200 μl totalwell volume; 4) PHOLASIN® stock, conc [10 ug/ml] was prepared by adding5 ml assay buffer to vial (50 μg PHOLASIN® from mollusca, KnightScientific) and stored frozen; 50 μl per assay; 5) XO stock, conc [˜676mU XO/ml] was prepared by pipetting 40 μl stock (XO from bovine milk,Sigma) to 2 ml assay buffer and stored refrigerated; 40 μl per assay; 6)PNP stock, conc [˜701 mU PNP/ml] was prepared by pipetting 75 μl stock(PNP from human RBC, Sigma) to 2 ml assay buffer and store refrigerated;40 μl per assay.

For Table 7: 1) Stock xanthine [164.4 μM or 25 μg/ml] in DI. wasprepared by adding 25 mg in 1000 mL DI (or assay buffer); 2) Workingstock (WS) WS-1 (16.4 μM) 100 μl stock xanthine (1:10 stock) 900 μlassay buffer; 3) Final xanthine conc based on 200 μl total well volume;4) PHOLASIN® conc [10 μg/ml] was prepared by adding 5 ml assay buffer tovial (50 μg PHOLASIN® from mollusca, Knight Scientific) and storedfrozen; 50 ml per assay; 5) XO conc stock [˜676 mU XO/ml] was preparedby pipetting 40 μl stock (XO from bovine milk, Sigma) to 2 ml assaybuffer and stored refrigerated; 40 μl per assay.

For Table 8: 1) Stock hypoxanthine [183.7 μM or 25 μg/ml] in DI wasprepared by adding 25 mg in 1000 mL DI (or assay buffer); 2) Workingstock (WS) WS-1 (18.37 μM) 100 μl stock hypoxanthine (1:10 stock) 900 μlassay buffer WS-2 (1.84 μM) 100 μl WS-1 hypoxanthine (1:10 WS-1) 900 μlassay buffer; 3) Final total hypoxanthine conc based on 200 μl totalwell volume; 4) PHOLASIN® conc [10 μg/ml] was prepared by adding 5 mlassay buffer to vial (50 μg PHOLASIN® from mollusca, Knight Scientific)and stored frozen; 50 μl per assay; 5) XO stock, conc [˜676 mU XO/ml]was prepared y pipetting 40 μl stock (XO from bovine milk, Sigma) to 2ml assay buffer and stored refrigerated; 40 μl per assay; 6) Targetrange of nucleoside/purine assay (includes xanthine plus inoaine andhypoxanthine conversion to xanthine) is ˜2 μM (normals) up to ˜100 μM(ischemic); 7) Sensitivity and linearity of the nucleoside/purine assay(if 1:10 dilution of plasma) needs to be ˜0.1 up to ˜10 μM.

TABLE 6 Typical spreadsheet used for inosine luminescence experiments.Final Inosine Inosine Standard Inosine Assay buffer Conc. [uM] (ul) WS[uM] (ul) 0.0 0 0 70.0 1.0 21.5 9.32 48.5 2.5 53.6 9.32 16.4 5.0 10.793.2 59.3 10.0 21.5 93.2 48.5 20.0 42.9 93.2 27.1 30.0 64.4 93.2 5.6

TABLE 7 Typical spreadsheet used for xanthine luminescence experiments.Final Xanthine Xanthine Standard Xanthine Assay buffer Conc. [uM] (ul)WS [uM] (ul) 0.0 0 0 110.0 1.0 12.2 16.4 97.8 2.5 30.5 16.4 79.5 5.061.0 16.4 49.0 10.0 12.2 164.4 97.8 20.0 24.3 164.4 85.7 30.0 36.5 164.473.5

TABLE 8 Typical spreadsheet used for hypoxanthine luminescenceexperiments. Final Standard Hypoxanthine Hypoxanthine Hypoxanthine Assaybuffer Conc. [uM] (ul) WS [uM] (ul) 0.0 0 0 110.0 0.1 10.9 1.84 99.1 0.221.8 1.84 88.2 0.5 54.4 1.84 55.6 1.0 10.9 183.7 99.1 2.0 21.8 183.788.2 5.0 5.4 183.7 104.6 10.0 10.9 183.7 99.1

Luminescence Computations

All computations were performed using BMG Excel software with built inmacros to carry out basic computations (e.g. additions, subtractions,etc.) and regression analysis (for standard curves, etc.) and dataprocessing set points as defined by the method. FIGS. 10 and 11represent a scan (FIG. 10, plasma with 10 μM hypoxanthine) and the RLUtabulated results (e.g. BMG Excel® Table 1, 2, and 3 in FIG. 11) fromraw data acquired over the analytical run and with data acquisition setat one data point per second. The background (baseline) luminescencesignal (labeled as Range 1 and presented in BMG Excel® Table 1 of FIG.11) can be caused by reagents (e.g. buffer, PHOLASIN®, PNP, plasma) andelectronic noise and was calculated as the maximum RLU signal betweenscan times 100-118 seconds. It would have been preferable to average thebackground RLU signal; however the BMG Excel® software was written tohave the same computation applied to both table ranges and does notcurrently allow the flexibility of independent computations on eachindividual table.

The peak luminescence signal from the generation of light from PHOLASIN®(labeled as Range 2 and presented in BMG Excel® Table 2 of FIG. 11) andsuperoxide anion radicals was calculated as the maximum RLU peak heightsignal between scan times 119-222 seconds. BMG Excel® Table 3 (FIG. 11)represents the net RLU and is calculated by subtracting the backgroundsignal (BMG Excel® Table 1 of FIG. 11) from the peak luminescent signal(BMG Excel® Table 2, FIG. 11). The use of the peak height response ofthe RLU was used for the computations on these plasma samples, as somepatient plasma samples RLU responses were very slow to return tobackground (baseline) RLU levels. The cause of the slow RLU signalreturn to baseline is unknown, but may be due to patient medications(e.g. vasodilators, salicylic acid) used for treatment of acute MIpatients.

This luminescence method was developed to compare the RLU differencesbetween healthy normal individual plasma samples (negative control) andsamples from ED non-traumatic chest pain patients that may beexperiencing acute cardiac ischemia. A comparison was made of the netRLU value between the non-traumatic chest pain patient and negativecontrol sample, using a calculated 99% RLU reference cut-off valuegenerated from healthy normal individuals, as the decision making RLUcut-off level. Determining the 99% RLU cut-off value is best bedetermined using a large number of healthy normal individuals(e.g. >>100) and calculated using the RLU mean value plus the 2.326standard deviations (a=0.01, one tail, 99% confidence interval), andwould be used to determine whether the patient has acute cardiacischemia causing the reported chest pain. For example, if anon-traumatic chest pain patient net RLU was similar to a negativecontrol sample net RLU, then the patient was most likely not having anacute cardiac ischemic event, but had some other type of medicalcondition (e.g. anxiety, heartburn) causing the reported chest pain.However, if a patient's net RLU was above the 99% RLU reference cut-offvalue for healthy normal individuals, then the patient was probablyexperiencing an acute cardiac ischemic event, and would requireimmediate medical attention, as it may lead to acute MI and potentialadverse outcome.

Results and Discussion

To setup the new luminometer equipment, a standardized plate modeluminescence test kit was purchased (ABEL 61M Antioxidant Test Kit,Knight Scientific, Ltd) which evaluates antioxidant capability usingxanthine/xanthine oxidase and PHOLASIN®. This test kit was used toqualify the new luminometer equipment using a standardized plate mode(glow technique). However, method modifications were necessary as theplate mode analysis run time was approximately 30 min and had lowsensitivity (FIG. 12) as it is developed primarily for antioxidant andglow kinetic type studies, which would be insufficient for the objectiveof a rapid and sensitive assay.

Adjustments were made to the level of XO used for analysis to increasethe reaction rate (flash mode) and the incubation time of PNP enzyme forplasma inosine conversion to hypoxanthine. The starting level of XOenzyme level for the plate mode was approximately 10.25 mU/ml afterreconstitution with assay buffer. With adjustment of XO to increase theconcentration, the final working concentration was approximately 676mU/ml. This resulted in an analysis time reduction from approximately 30min to 5 min (FIG. 13). Since the commercial kit from Knight Scientific(plate mode) was set up for xanthine/xanthine oxidase analysis andstudies on material antioxidant capabilities, it was necessary toincrease the XO level to additionally incorporate plasma hypoxanthinelevels, but more importantly to reduce the time of analysis to under 10min (i.e. switch from glow mode to flash mode kinetics).

A standard curve of hypoxanthine was evaluated at concentrations from2.3 to 30.3 μM and demonstrated sufficient linearity (normal linearregression) with correlation coefficient >0.9990 (n=2). The incubationtime of purine nucleoside phosphorylase was evaluated at 60 and 120second equilibration times using 10 μM inosine as the substrate with themonitoring of hypoxanthine level (FIGS. 14A-B). Therefore, the PNPincubation time should remain set at 120 sec to allow for completeinosine to hypoxanthine conversion, with subsequent XO injection tostart the luminescence reaction. In some embodiments, in order to reducethe overall analysis time, the PNP enzyme is added to the samplecollection tube (e.g. BD vacuutainer), with inosine conversion thenoccurring during the whole blood to plasma centrifugation step,eliminating the need for a 120 sec PNP incubation time; and reducing theanalysis time to only 30 seconds (assumes injection of XO at 0.1 sec andmeasurement of peak height RLU response).

A study of the effect of plasma uric acid on luminescence response wasperformed. Since uric acid is found in plasma at relatively higherconcentrations (normal range ˜350-475 μM) and is a known antioxidant, itwas important to evaluate its potential effect on the luminescencesignal. As seen in FIG. 15A, the uric acid's antioxidant affectsdecreases the luminescence signal (˜50% quenching). To address the uricacid, an experiment was performed using strong anion exchange (SAX)resin to remove organic anions from the plasma matrix. Also seen in FIG.13B is a 1:100 dilution of plasma and subsequent use of the SAX pipettip (Varian, Inc, CA, USA); both demonstrated that removal of potentialinterfering organic acids (e.g. urate at pH 7.4) resulted with anincrease in luminescence response and sensitivity. Since the blankplasma used had approximately 500 nM hypoxanthine, the 1:100 dilutionusing deionized water and subsequent use of SAX sorbent makes detectionlevels of hypoxanthine at the pM levels attainable.

A second approach to eliminate the uric acid was to utilize uricase(˜1.1 U/ml from Arthrobacter globiformis bacteria, Sigma, USA) duringthe PNP incubation time in an attempt to eliminate the endogenous uricacid. As seen in FIG. 16, it appears that the XO enzyme is deactivated(product inhibition) by the presence of large amounts of by-producthydrogen peroxide that is generated by uricase activity. As oneby-product of XO activity is the production of hydrogen peroxide, thisfinding was not completely surprising due to the effects of productinhibition on XO enzyme turnover. One possible solution to eliminate thegenerated hydrogen peroxide is to use horseradish peroxidase, whichcatalyzes hydrogen peroxide to products water and oxygen, but thisadditional enzyme would only add to the complexity and cost of theanalysis and therefore not evaluated. However, the use of the bothuricase and SAX pipet tip technology to eliminate organic acids (e.g.uric acid) was probably not necessary to use, as differences inluminescence response between the healthy normal individuals andconfirmed acute MI patients (elevated cTnT levels) was significant(t-test, a=0.05, p<0.01), when using the 99% percentile (a=0.01, onetail, 2.326 standard deviations for n=6) as the calculated biomarkercut-off reference value for acute cardiac ischemia (FIG. 17). Eventhough this research utilized a small sample set for evaluation (n=6 foreach group), the 99% cut-off for healthy normal individuals was 5,946RLU, with all six cTnT patients clearly above this calculated decisionpoint cut-off RLU level. The luminescence method was optimized for rapidevaluation of hypoxanthine in plasma to potentially be used in an EDclinical type environment. For this research study, method parameterssuch as calibration, repeatability and limit of detection were evaluatedusing hypoxanthine standards. The method demonstrated linearity from2.3-30.3 μM hypoxanthine in assay buffer (R=0.9990, n=2) (FIG. 18). Thisrange covered the low and midpoint total hypoxanthine concentrations ofsamples from HPLC analysis of healthy individuals and non-traumaticchest pain patients, and focused on the potential biomarker cut-offconcentration for this small group of samples (n=20). Repeatability(n=3) was evaluated by fortification of plasma at basal (˜0.5 μM) and1.5 μM hypoxanthine concentrations (final well levels) and demonstratedby consistent RLU overlays (FIG. 19A-B).

Conclusion

A rapid luminescence method was developed for the detection of inosineand hypoxanthine in human plasma. Using only 20 ul of plasma (heparin)and instrument direct injectors, the method allowed for the rapid (<5min) detection of total hypoxanthine (as inosine is converted tohypoxanthine using enzyme PNP) concentrations, which may be used as abiomarker of acute cardiac ischemia. The use of a hypothetical cut-offlevel (e.g. 99% confidence) relative luminescence unit (RLU) fordecision making (i.e. positive level, negative level) may be the mosteffective use of this rapid screening assay. The method was utilized forevaluation of plasma samples from healthy individuals and cardiacpatients with confirmed acute myocardial infarction (hospital documentedelevated plasma cTnT levels), and demonstrated the potential of thisrapid assay to be used as a diagnostic tool, for use by emergencydepartment services personnel on non-traumatic chest pain patientssuspected of undergoing acute cardiac ischemia.

Example 4 Use of the Assay of the Invention in the Evaluation ofPatients with Chest Pain and the Diagnosis or Ruling Out of a CardiacIschemic Event

In the United States alone, approximately 8 million patients areevaluated in emergency rooms due to non-traumatic chest pain. It isestimated that about 2-5% of these patients are actually experiencingacute myocardial infarction, but are mis-diagnosed and discharged. Thiscould be, for example, about 400,000 people annually that areincorrectly diagnosed and discharged without proper therapy. World-wide,the numbers are even more daunting. Possible other conditions that maycause chest pain include gastroesophageal reflux, acid reflux, variousmusculoskeletal spasms, pneumonia, aortic dissection, nerve impingement,pulmonary emboli, spontaneous pneumothorax, acute pericarditis,heartburn, asthma, anxiety and ulcers. Typical testing for diagnosingacute MI includes noting patient's vital signs, symptoms and history; anelectrocardiogram (ECG) which measures heart electrical activity; ablood analysis, which is limited to detecting indicators of heat tissuenecrosis (e.g. cardiac troponin I and T, myoglobin, creatine kinase); anechocardiogram, which can show both false positive and false negativeresults, up to about 20%; and stress testing (e.g. on a treadmill),which is rarely done. The primary goal of an emergency department is torule-in myocardial infarction, which requires immediate treatment. TheWorld Health Organization recommends that 2 out of 3 possible indicatorsbe positive in order to diagnose probably MI. Ruling out MI, however, isextremely difficult and using the technology available prior to thepresent invention, could take several days. As many as ⅓ of patientshave few or no classical symptoms. Significantly, a majority of acutemyocardial infarction (AMI) deaths occur within a 12-hour window ofonset but cannot be prevented without proper diagnosis.

FIG. 20 is a flow chart of a typical ER triage of a patient presentingwith chest pain. However, this chart includes the detection of the earlybiomarkers inosine and hypoxanthine, using the rapid luminescence testdescribed herein. Using the method, within minutes of obtaining a bloodsample, an accurate indicator of cardiac ischemia can be obtained. Iflevels of inosine and/or hypoxanthine are elevated (e.g. confidencelevel of 99% or greater), then an assumption of early cardiac ischemiacan be made and appropriate treatment to prevent AMI can be instigatedimmediately, particularly if one other test is also positive.Thereafter, the test may be run again as necessary or advisable, e.g. toconfirm that intervention has succeeded, to test for a possible secondAMI, etc. Even if the test is negative, a repeat assay after arelatively short time period of observation (e.g. about 1-3 hours), andthereafter as deemed necessary, can be carried out to confirm thatindeed an AMI is not likely to be imminent, and another cause of thechest pain should be explored. Thus, the percentage of patients who aremisdiagnosed and discharged without appropriate treatment is minimized.

While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

1. A method of diagnosing an ischemic event in a patient, comprising thesteps of adding xanthine oxidase (XO) enzyme to a biological sampleobtained from said patient; measuring metabolic byproducts of enzymaticactivity of XO enzyme on xanthine and hypoxanthine in said biologicalsample; and determining whether or not said patient is experiencing anischemic event based on a measurement made in said measuring step. 2.The method of claim 1 wherein said steps of adding, measuring, anddetermining are performed in 10 minutes or less.
 3. The method of claim1, wherein said steps of adding, measuring, and determining areperformed in 1 minute or less.
 4. The method of claim 1 wherein saidmetabolic byproducts are selected from the group consisting of hydrogenperoxide, superoxide anion radicals (SAR), and hydroxyl free radicals.5. The method of claim 1 wherein if said measurement in relativeluminescent units is equivalent to a measured value of 4.6 μM or greaterfor inosine, hypoxanthine and xanthine in said biological sample, thensaid determining step determines that said patent is having an ischemicevent.
 6. The method of claim 1 wherein said measuring step includes thestep of using a chemiluminescent agent in said biological sample whenmeasuring said metabolic byproducts.
 7. The method of claim 6, whereinsignal enhancers are used together with said chemiluminescent agent. 8.The method of claim 1 wherein said measuring step includes using asubstrate to which said XO enzyme is bound or associated.
 9. The methodof claim 7 wherein said substrate is selected from the group consistingof a test strip and a bead.
 10. The method of claim 1 further comprisingthe step of decreasing the antioxidant effect of uric acid in saidbiological sample to increase sensitivity during said measuring step.11. The method of claim 1 further comprising the step of diluting saidbiological sample prior to said measuring step.
 12. The method of claim11 wherein said step of diluting includes the step of using a diluentwhich buffers said biological sample between pH 7.2 and pH 7.8.
 13. Themethod of claim 1, further comprising the step of adding purinenucleoside phosphorylase (PNP) to said sample prior to said step ofadding XO.
 14. The method of claim 1, wherein said ischemic event isassociated with a condition selected from the group consisting ofcardiac ischemia, angina, stroke, and acute coronary syndrome.
 15. Themethod of claim 1, wherein said biological sample is selected from thegroup consisting of blood, plasma, saliva, spinal fluid, and urine. 16.A method of diagnosing an ischemic event in a patient, comprising thesteps of indirectly detecting, using a chemiluminescent label, a levelof one or more of inosine, xanthine, and hypoxanthine in said biologicalsample; and determining whether or not said patient is experiencing anischemic event based on a measurement made in said detecting step. 17.The method of claim 16, further comprising the step of adding PNP to abiological sample obtained from said patient,
 18. The method of claim17, wherein said step of indirectly detecting is carried out two minutesafter said step of adding PNP.
 19. A method of diagnosing an ischemicevent in a patient, comprising the steps of: detecting, usingantibodies, the level of one or more of inosine or hypoxanthine in abiological sample; and determining whether or not said patient isexperiencing an ischemic event based on a measurement made in saiddetecting step.
 20. A device for diagnosing an ischemic event in apatient, comprising: a substrate with one or more enzymes selected frompurine nucleoside phosphorylase (PNP) and xanthine oxidase (XO), and aluminescent material associated therewith on which a blood or plasmasample is deposited; and a detector for measuring luminescence on saidsubstrate wherein measurements made with said detector are equated towhether or not a patient is experiencing an ischemic event.
 21. Thedevice of claim 20, wherein said detector includes a photodiode orphotomultiplier.